Anti-LGR5 antibodies and immunoconjugates

ABSTRACT

The invention provides anti-LgR5 antibodies and immunoconjugates and methods of using the same.

This application claims the benefit of U.S. Provisional Application No.61/618,232, filed Mar. 30, 2012; U.S. Provisional Application No.61/683,048, filed Aug. 14, 2012; and U.S. Provisional Application No.61/778,710, filed Mar. 13, 2013; each of which is incorporated byreference herein in its entirety for any purpose.

FIELD OF THE INVENTION

The present invention relates to anti-LgR5 antibodies andimmunoconjugates and methods of using the same.

BACKGROUND

Leucine-rich repeat-containing G protein-coupled receptor 5 (LgR5) is aseven-transmembrane protein found on the surface of actively cyclingintestinal stem cells (ISCs). LgR5-expressing ISCs are sensitive to Wntmodulation and are primarily responsible for homeostatic regeneration ofthe intestinal epithelium. Elimination of LgR5-expressing cells in micedoes not affect homeostasis of intestinal epithelium, however,suggesting that other cell types can compensate for loss of this cellpopulation. Tian et al., Nature 478: 255-259 (2011). R-spondins enhanceWNT signaling by WNT3A, and all four R-spondins, RSPO1, RSPO2, RSPO3,and RSPO4, are able to bind to LgR5. Lau et al., Nature 476: 293-297(2011).

Human LgR5 is a 907 amino acid protein, of which ˜540 amino acids arepredicted to be in the extracellular space following cleavage of theamino-terminal signal sequence. LgR5 comprises 17 imperfect leucine-richrepeat motifs in the ectodomain, and a cysteine-rich region locatedbetween the leucine-rich repeats and the first transmembrane domain.

There is a need in the art for agents that target LgR5 for the diagnosisand treatment of LgR5-associated conditions, such as cancer. Theinvention fulfills that need and provides other benefits.

SUMMARY

The invention provides anti-LgR5 antibodies and immunoconjugates andmethods of using the same.

In some embodiments, an isolated antibody that binds to LgR5 isprovided. In some embodiments, the antibody has at least one or more ofthe following characteristics, in any combination: (a) binds to anepitope within amino acids 22-555 of SEQ ID NO: 67 and/or binds to anepitope within amino acids 22-123 of SEQ ID NO: 67 and/or binds to anepitope within amino acids 22-323 of SEQ ID NO: 67 and/or binds to anepitope within amino acids 22-424 of SEQ ID NO: 67 and/or binds to anepitope within amino acids 324-555 of SEQ ID NO: 67 and/or binds to anepitope within amino acids 324-424 of SEQ ID NO: 67; (b) binds LgR5 withan affinity of ≦5 nM, or ≦4 nM, or ≦3 nM, or ≦2 nM, or ≦1 nM, andoptionally ≧0.0001 nM, or ≧0.001 nM, or ≧0.01 nM; (c) does notsignificantly disrupt the binding of R-spondin (RSPO) to LgR5; (d) doesnot significantly disrupt beta-catenin signaling; (e) does notsignificantly disrupt RSPO activation of LgR5 signaling; (f) activatescaspase 3 cleavage; (g) recognizes both human and rodent LgR5; (h)recognizes human LgR5 but not rodent LgR5; (i) does not significantlyinhibit tumor growth in its unconjugated form; and (j) does not inducestem cell differentiation.

In some embodiments, the isolated anti-LgR5 antibody binds to an epitopewithin amino acids 22-323 of SEQ ID NO: 67 with an affinity of ≦5 nM, or≦4 nM, or ≦3 nM, or ≦2 nM, or ≦1 nM, and optionally ≧0.0001 nM, or≧0.001 nM, or ≧0.01 nM.

In some embodiments, the isolated anti-LgR5 antibody binds to an epitopewithin amino acids 22-123 of SEQ ID NO: 67 with an affinity of ≦5 nM, or≦4 nM, or ≦3 nM, or ≦2 nM, or ≦1 nM, and optionally ≧0.0001 nM, or≧0.001 nM, or ≧0.01 nM.

In some embodiments, the isolated anti-LgR5 antibody binds to an epitopewithin amino acids 324-424 of SEQ ID NO: 67 with an affinity of ≦5 nM,or ≦4 nM, or ≦3 nM, or ≦2 nM, or ≦1 nM, and optionally ≧0.0001 nM, or≧0.001 nM, or ≧0.01 nM.

In some embodiments of any of the isolated anti-LgR5 antibodies, theanti-LgR5 antibody does not significantly disrupt the binding ofR-spondin (RSPO) to LgR5. In some embodiments of any of the isolatedanti-LgR5 antibodies, the anti-LgR5 antibody does not significantlydisrupt wnt/beta-catenin signaling. In some embodiments of any of theisolated anti-LgR5 antibodies, the anti-LgR5 antibody does notsignificantly disrupt RSPO activation of LgR5 signaling. In someembodiments of any of the isolated anti-LgR5 antibodies, the anti-LgR5antibody activates caspase 3 cleavage. In some embodiments of any of theisolated anti-LgR5 antibodies, the anti-LgR5 antibody recognizes bothhuman and rodent LgR5. In some embodiments of any of the isolatedanti-LgR5 antibodies, the anti-LgR5 antibody recognizes human LgR5 butnot rodent LgR5. In some embodiments of any of the isolated anti-LgR5antibodies, the anti-LgR5 antibody does not significantly inhibit tumorgrowth in its unconjugated form. In some embodiments of any of theisolated anti-LgR5 antibodies, the anti-LgR5 antibody does not inducestem cell differentiation.

In some embodiments of any of the isolated anti-LgR5 antibodies, theanti-LgR5 antibody is a monoclonal antibody. In some embodiments of anyof the isolated anti-LgR5 antibodies, the anti-LgR5 antibody is a human,humanized, or chimeric antibody. In some embodiments of any of theisolated anti-LgR5 antibodies, the anti-LgR5 antibody antibody is anIgG1, IgG2a or IgG2b antibody. In some embodiments of any of theisolated anti-LgR5 antibodies, the anti-LgR5 antibody is an antibodyfragment that binds LgR5. In some embodiments of any of the isolatedanti-LgR5 antibodies, LgR5 is human LgR5 of SEQ ID NO: 67.

In some embodiments, an antibody that binds LgR5 binds an epitope withinamino acids 22-323 of SEQ ID NO: 67. In some embodiments, the antibodybinds to LgR5 with an affinity of ≦5 nM. In some embodiments, theantibody is a monoclonal antibody. In some embodiments, the antibody isa human, humanized, or chimeric antibody. In some embodiments, theantibody is an IgG1, IgG2a or IgG2b antibody. In some embodiments, theantibody is an antibody fragment that binds LgR5. In some embodiments,LgR5 is human LgR5 of SEQ ID NO: 67.

In some embodiments, the antibody comprises (a) HVR-H3 comprising theamino acid sequence of SEQ ID NO: 32, (b) HVR-L3 comprising the aminoacid sequence of SEQ ID NO: 29, and (c) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 31. In some embodiments, the antibody comprises(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 30, (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 31, and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 32. In someembodiments, the antibody further comprises a heavy chain framework FR3sequence of SEQ ID NO: 41. In some embodiments, the antibody furthercomprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:27, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 28, and(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29. In someembodiments, an isolated antibody comprises (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 27, (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 28, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 29. In some embodiments, the antibody furthercomprises a light chain framework FR3 sequence of SEQ ID NO: 35.

In some embodiments, an isolated antibody comprises (a) a VH sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO: 8; or (b) a VL sequence having at least 95% sequence identity tothe amino acid sequence of SEQ ID NO:7; or (c) a VH sequence as in (a)and a VL sequence as in (b). In some embodiments, the antibody comprisesa VH sequence of SEQ ID NO: 8. In some embodiments, the antibodycomprises a VL sequence of SEQ ID NO: 7. In some embodiments, andisolated antibody comprises a VH sequence of SEQ ID NO: 8 and a VLsequence of SEQ ID NO: 7.

In some embodiments, an antibody that binds LgR5 comprises (a) HVR-H3comprising the amino acid sequence of SEQ ID NO: 56, (b) HVR-L3comprising the amino acid sequence of SEQ ID NO: 53, and (c) HVR-H2comprising the amino acid sequence of SEQ ID NO: 55. In someembodiments, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 54, (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 55, and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO: 56. In some embodiments, the antibody further comprises (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51, (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 52, and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 53. In someembodiments, an isolated antibody comprises (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 51, (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 52, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 53.

In some embodiments, an isolated antibody comprises (a) a VH sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO: 24; or (b) a VL sequence having at least 95% sequence identity tothe amino acid sequence of SEQ ID NO:23; or (c) a VH sequence as in (a)and a VL sequence as in (b). In some embodiments, the antibody comprisesa VH sequence of SEQ ID NO: 24. In some embodiments, the antibodycomprises a VL sequence of SEQ ID NO: 23. In some embodiments, andisolated antibody comprises a VH sequence of SEQ ID NO: 24 and a VLsequence of SEQ ID NO: 23.

In some embodiments, an antibody that binds LgR5 comprises (a) HVR-H3comprising the amino acid sequence of SEQ ID NO: 50, (b) HVR-L3comprising the amino acid sequence of SEQ ID NO: 47, and (c) HVR-H2comprising the amino acid sequence of SEQ ID NO:495. In someembodiments, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 48, (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 49, and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO: 50. In some embodiments, the antibody further comprises (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 45, (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 46, and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 47. In someembodiments, an isolated antibody comprises (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 45, (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 46, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 47.

In some embodiments, an isolated antibody comprises (a) a VH sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO: 22; or (b) a VL sequence having at least 95% sequence identity tothe amino acid sequence of SEQ ID NO:21; or (c) a VH sequence as in (a)and a VL sequence as in (b). In some embodiments, the antibody comprisesa VH sequence of SEQ ID NO: 22. In some embodiments, the antibodycomprises a VL sequence of SEQ ID NO: 21. In some embodiments, andisolated antibody comprises a VH sequence of SEQ ID NO: 22 and a VLsequence of SEQ ID NO: 21.

In some embodiments, an antibody that binds LgR5 comprises (a) HVR-H3comprising the amino acid sequence of SEQ ID NO: 62, (b) HVR-L3comprising the amino acid sequence of SEQ ID NO: 59, and (c) HVR-H2comprising the amino acid sequence of SEQ ID NO: 61. In someembodiments, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 60, (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 61, and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO: 62. In some embodiments, the antibody further comprises (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 57, (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 58, and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 59. In someembodiments, an isolated antibody comprises (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 57, (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 58, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 59.

In some embodiments, an isolated antibody comprises (a) a VH sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO: 26; or (b) a VL sequence having at least 95% sequence identity tothe amino acid sequence of SEQ ID NO:25; or (c) a VH sequence as in (a)and a VL sequence as in (b). In some embodiments, the antibody comprisesa VH sequence of SEQ ID NO: 26. In some embodiments, the antibodycomprises a VL sequence of SEQ ID NO: 25. In some embodiments, andisolated antibody comprises a VH sequence of SEQ ID NO: 26 and a VLsequence of SEQ ID NO: 25.

In some embodiments, an isolated nucleic acid that encodes an antibodydescribed herein is provided. In some embodiments, a host cellcomprising the nucleic acid is provided. In some embodiments, a methodof producing an antibody described herein is provided. In someembodiments, the method comprises culturing the host cell comprising thenucleic acid that encodes an antibody.

In some embodiments, immunoconjugates are provided. In some embodiments,an immunoconjugate comprises an anti-LgR5 antibody and a cytotoxicagent. In some embodiments, an immunoconjugate has the formulaAb-(L-D)p, wherein: (a) Ab is an antibody described herein; (b) L is alinker; (c) D is a drug selected from a maytansinoid, an auristatin, acalicheamicin, a pyrrolobenzodiazepine, and a nemorubicin derivative;and (d) p ranges from 1-8. In some embodiments, D is an auristatin. Insome such embodiments, D has formula D_(E)

wherein R² and R⁶ are each methyl, R³ and R⁴ are each isopropyl, R⁵ isH, R⁷ is sec-butyl, each R⁸ is independently selected from CH₃, O—CH₃,OH, and H; R⁹ is H; and R¹⁸ is —C(R⁸)₂—C(R⁸)₂— aryl. In someembodiments, D is MMAE having the structure:

In some embodiments, D is a pyrrolobenzodiazepine of Formula A:

wherein the dotted lines indicate the optional presence of a double bondbetween C1 and C2 or C2 and C3; R² is independently selected from H, OH,═O, ═CH₂, CN, R, OR, ═CH—R^(D), ═C(R^(D))₂, O—SO₂—R, CO₂R and COR, andoptionally further selected from halo or dihalo, wherein R^(D) isindependently selected from R, CO₂R, COR, CHO, CO₂H, and halo; R⁶ and R⁹are independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo; R⁷ is independently selected from H, R, OH, OR, SH,SR, NH₂, NHR, NRR′, NO₂, Me₃Sn and halo; Q is independently selectedfrom O, S and NH; R¹¹ is either H, or R or, where Q is O, SO₃M, where Mis a metal cation; R and R′ are each independently selected fromoptionally substituted C₁₋₈ alkyl, C₁₋₁₂ alkyl, C₃₋₈ heterocyclyl, C₃₋₂₀heterocyclyl, and C₅₋₂₀ aryl groups, and optionally in relation to thegroup NRR′, R and R′ together with the nitrogen atom to which they areattached form an optionally substituted 4-, 5-, 6- or 7-memberedheterocyclic ring; R¹², R¹⁶, R¹⁹ and R¹⁷ are as defined for R², R⁶, R⁹and R⁷ respectively; R″ is a C₃₋₁₂ alkylene group, which chain may beinterrupted by one or more heteroatoms and/or aromatic rings that areoptionally substituted; and X and X′ are independently selected from O,S and N(H). In some such embodiments, D is

wherein n is 0 or 1.

In some embodiments, D is a nemorubicin derivative. In some embodiments,D has a structure selected from:

In some embodiments, an immunoconjugate comprises a linker that iscleavable by a protease. In some embodiments, the linker comprises aval-cit dipeptide or a Phe-Lys dipeptide. In some embodiments, animmunoconjugate comprises a linker that is acid-labile. In some suchembodiments, the linker comprises hydrazone.

In some embodiments, an immunoconjugate has a formula selected from:

wherein S is a sulfur atom;

In some embodiments, p ranges from 2-5.

In some embodiments, an immunoconjugate comprises an antibody thatcomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:30, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 31, and(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 32; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 27, (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 28, and (0 HVR-L3comprising the amino acid sequence of SEQ ID NO: 29. In someembodiments, an immunoconjugate comprises an antibody that comprises aVH sequence of SEQ ID NO: 8 and a VL sequence of SEQ ID NO: 7.

In some embodiments, pharmaceutical formulations are provided. In somesuch embodiments, a pharmaceutical formulation comprises animmunoconjugate comprising an antibody that binds LgR5, e.g., asdescribed herein. In some embodiments, a pharmaceutical formulationfurther comprises an additional therapeutic agent. In some embodiments,the additional therapeutic agent is Avastin® (bevacizumab).

In some embodiments, methods of treating individuals having LgR5positive cancers are provided. In some such embodiments, a methodcomprises administering a pharmaceutical formulation comprising animmunoconjugate comprising an antibody that binds LgR5, e.g., asdescribed herein. In some embodiments, the LgR5-positive cancer isselected from colorectal cancer, pancreatic cancer, ovarian cancer, andendometrial cancer. In some embodiments, the LgR5-positive cancer is asmall intestine cancer. In some embodiments, a small intestine cancer isa cancer of the duodenum, jejunum, and/or ilium. In some embodiments, asmall intestine cancer is a cancer of the jejunum and/or ilium. In someembodiments, an LgR5-positive cancer comprises a Kras mutation, an APCmutation, or both a Kras mutation and an APC mutation (e.g., in at leasta portion of the cancer cells). In some embodiments, a method comprisesadministering an additional therapeutic agent to the individual. In somesuch embodiments, the additional therapeutic agent is Avastin®(bevacizumab).

In some embodiments, methods of inhibiting proliferation of anLgR5-positive cell are provided. In some embodiments, the methodcomprising exposing the cell to an immunoconjugate comprising anantibody that binds LgR5 under conditions permissive for binding of theimmunoconjugate to LgR5 on the surface of the cell. In some embodiments,an antibody that binds LgR5 is an antibody described herein. In someembodiments, proliferation of the cell is thereby inhibited. In someembodiments, the cell is a colorectal, small intestine, pancreatic,ovarian, or endometrial cancer cell.

In some embodiments, an antibody that binds LgR5 is conjugated to alabel. In some embodiments, an antibody that binds LgR5 is an antibodydescribed herein. In some embodiments, the label is a positron emitter.In some embodiments, the positron emitter is ⁸⁹Zr.

In some embodiments, a method of detecting human LgR5 in a biologicalsample is provided. In some embodiments, a method comprises contactingthe biological sample with an anti-LgR5 antibody under conditionspermissive for binding of the anti-LgR5 antibody to a naturallyoccurring human LgR5, and detecting whether a complex is formed betweenthe anti-LgR5 antibody and a naturally occurring human LgR5 in thebiological sample. In some embodiments, an anti-LgR5 antibody is anantibody described herein. In some embodiments, the biological sample isa colorectal cancer sample, small intestine cancer sample, pancreaticcancer sample, ovarian cancer sample, or endometrial cancer sample.

In some embodiments, a method for detecting an LgR5-positive cancer isprovided. In some such embodiments, a method comprises (i) administeringa labeled anti-LgR5 antibody to a subject having or suspected of havingan LgR5-positive cancer, and (ii) detecting the labeled anti-LgR5antibody in the subject, wherein detection of the labeled anti-LgR5antibody indicates a LgR5-positive cancer in the subject. In someembodiments, an anti-LgR5 antibody is an antibody described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a graphic representation of the levels of human LgR5 geneexpression in various tissues, as described in Example A. The inset inFIG. 1 shows a graphic representation of the levels of human LgR5 geneexpression in normal colon tissues and colon tumors, as described inExample A.

FIG. 2 shows expression of LgR5 in colon tumors by in situhybridization, as described in Example B.

FIG. 3 shows (A) the prevalence of various levels of LgR5 expression ina colon tumor tissue microarray, and (B) the heterogeneity of LgR5expression in three cores from each colorectal adenocarcinoma sample,both determined by in situ hybridization, as described in Example B.

FIG. 4 shows the properties of certain anti-LgR5 monoclonal antibodiesdeveloped as described in Examples C through F.

FIG. 5 shows an alignment of the light chain variable region sequencesof murine antibody mu8E11 and humanized variants thereof (hu8E11.v1 tohu8E11.v8). The three hypervariable regions (HVRs: HVR1, HVR2, HVR3) areindicated by boxes in the sequences.

FIG. 6 shows an alignment of the heavy chain variable region sequencesof murine antibody mu8E11 and humanized variants thereof (hu8E11.v1 tohu8E11.v8). The three hypervariable regions (HVRs: HVR1, HVR2, HVR3) areindicated by boxes in the sequences.

FIG. 7 shows the light chain variable region sequences of murineantibodies 3G12 and 2H6. The three hypervariable regions (HVRs: HVR1,HVR2, HVR3) are indicated by boxes in the sequences.

FIG. 8 shows the heavy chain variable region sequences of murineantibodies 3G12 and 2H6. The three hypervariable regions (HVRs: HVR1,HVR2, HVR3) are indicated by boxes in the sequences.

FIG. 9 shows affinity measurements of chimeric antibody ch8E11 andvarious humanized variants, as described in Example E.

FIG. 10 shows the light chain variable region sequence of human antibodyYW353. The three hypervariable regions (HVRs: HVR1, HVR2, HVR3) areindicated by boxes in the sequences.

FIG. 11 shows the heavy chain variable region sequence of human antibodyYW353. The three hypervariable regions (HVRs: HVR1, HVR2, HVR3) areindicated by boxes in the sequences.

FIG. 12A-C show an alignment of LgR5 from human, cynomolgus monkey, rat,and mouse.

FIG. 13 shows that anti-LgR5 immunoconjugates demonstrate efficacy inLoVo colon cancer xenografts, as described in Example L.

FIG. 14 shows that anti-LgR5 immunoconjugates demonstrate efficacy inD5124 pancreatic cancer xenografts, as described in Example M.

FIG. 15 shows that huYW353-vcMMAE immunoconjugate demonstrates efficacyat 3, 6, and 12 mg/kg in D5124 pancreatic cancer xenografts, asdescribed in Example M.

FIG. 16 shows LgR5 mRNA expression in normal tissue and polyps fromcolons of AV and AKV mice, as described in Example N.

FIG. 17 shows survival of AKV mice administered anti-LgR5 antibody andanti-LgR5 antibody-drug conjugate have longer survival times thancontrol AKV mice, as described in Example N.

FIG. 18 shows percentage of tumor area that is positive for cleavedcaspase 3 in AKV mice administered a control ADC, an anti-LgR5 ADC, oran anti-LgR5 antibody, as described in Example N.

FIG. 19 shows AKV mice administered anti-LgR5 antibody-drug conjugatehave longer survival times than untreated AKV mice and AKV miceadministered gp120-ADC or anti-LgR5, as described in Example N.

FIG. 20 shows LgR5+ area in small intestine polyps and colon polyps inAKV LgR5^(DTR/+) mice, as described in Example N.

FIG. 21 shows (A) CC3+GFP+ area per cellular area in control ADC andanti-LgR5-ADC treated AKV LgR5^(DTR/+) mice, and (B) exemplaryimmunohistochemistry staining in the control ADC and anti-LgR5-ADCtreated AKV LgR5^(DTR/+) mice, as described in Example N.

FIG. 22 shows Ki67+ area per cellular area (either GFP+ cells or GFP−cells) in control ADC and anti-LgR5-ADC treated AKV LgR5^(DTR/+) mice,as described in Example N.

FIG. 23 shows the ratio of GFP intensity to GFP+ area in crypts andtumors of AKV LgR5^(DTR/+) mice, as described in Example N.

FIG. 24 shows that huYW353-vcMMAE, hu8E11v2-vcMMAE, and ch8E11-vcMMAEimmunoconjugate demonstrates efficacy in D5124 pancreatic cancerxenografts, as described in Example O.

FIG. 25 shows that hu8E11v2-vcMMAE immunoconjugate demonstrates efficacyin D5124 pancreatic cancer xenografts, as described in Example O.

FIG. 26 shows that huYW353-vcMMAE and hu8E11v2-vcMMAE immunoconjugatesdemonstrate efficacy in LoVoX1.1 colon cancer xenografts, as describedin Example P.

FIG. 27 shows that hu8E11v2-vcMMAE immunoconjugate demonstrates efficacyin LoVoX1.1 colon cancer xenografts, as described in Example P.

FIG. 28 shows that huYW353-vcMMAE, huYW353-acetal-PNU, and huYW353-vcPNUimmunoconjugates demonstrate efficacy in D5124 pancreatic cancerxenografts, as described in Example Q.

FIG. 29 shows that hu8E11v2-acetal-PNU, hu8E11v2-vcPNU, and hu8E11v2-PNUimmunoconjugates demonstrate in D5124 pancreatic cancer xenografts, asdescribed in Example R.

FIG. 30 shows the results of administering certain hu8E11v2immunoconjugates and control antibody immunoconjugates in LoVoX1.1 coloncancer xenografts, as described in Example S.

FIG. 31 that hu8E11v2-acetal-PNU immunoconjugate demonstrates efficacyin LoVoX1.1 colon cancer xenografts in mice coadministered excesscontrol antibody, as described in Example S.

FIG. 32 shows that an anti-LgR5 huYW353 PBD immunoconjugate demonstratesefficacy in D5124 pancreatic cancer xenografts, as described in ExampleT.

FIG. 33 shows that an anti-LgR5 hu8E11v2 PBD immunoconjugate demonstrateefficacy in D5124 pancreatic cancer xenografts, as described in ExampleT.

FIG. 34 shows that an anti-LgR5 hu8E11v2 PBD immunoconjugatedemonstrates efficacy in a LoVoX1.1 colon cancer xenograft, as describedin Example U.

FIG. 35 shows the structures of (A) an antibody-vcMMAE immunoconjugate,(B) an antibody-acetal-PNU immunoconjugate, (C) an antibody-acetal-PNUimmunoconjugate, (D) an antibody-PNU immunoconjugate, and (E) anantibody-vcPBD immunoconjugate.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION I.Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The terms “anti-LgR5 antibody” and “an antibody that binds to LgR5”refer to an antibody that is capable of binding LgR5 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting LgR5. In one embodiment, the extent ofbinding of an anti-LgR5 antibody to an unrelated, non-LgR5 protein isless than about 10% of the binding of the antibody to LgR5 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to LgR5 has a dissociation constant (Kd) of ≦1 μm, ≦100 nM,≦10 nM, ≦5 Nm, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001nM (e.g., 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to10⁻¹³ M). In certain embodiments, an anti-LgR5 antibody binds to anepitope of LgR5 that is conserved among LgR5 from different species.

The term “antibody” is used herein in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody and that bindsthe antigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer,hepatoma, breast cancer, colon cancer, colorectal cancer, smallintestine cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, leukemia and otherlymphoproliferative disorders, and various types of head and neckcancer.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “epitope” refers to the particular site on an antigen moleculeto which an antibody binds.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The term “glycosylated forms of LgR5” refers to naturally occurringforms of LgR5 that are post-translationally modified by the addition ofcarbohydrate residues.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH(H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated antibody” is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated nucleic acid” refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-LgR5 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “LgR5,” as used herein, refers to any native, mature LgR5 whichresults from processing of an LgR5 precursor protein in a cell. The termincludes LgR5 from any vertebrate source, including mammals such asprimates (e.g. humans and cynomolgus monkeys) and rodents (e.g., miceand rats), unless otherwise indicated. The term also includes naturallyoccurring variants of LgR5, e.g., splice variants or allelic variants.The amino acid sequence of an exemplary human LgR5 precursor protein,with signal sequence (amino acids 1-21) is shown in SEQ ID NO: 67. Theamino acid sequence of an exemplary mature human LgR5 is shown in SEQ IDNO: 68. The predicted sequence for amino acids 33 to 907 of an exemplarycynomolgus monkey LgR5 is shown in SEQ ID NO: 69. The amino acidsequences for exemplary rat LgR5 precursor (with signal sequence, aminoacids 1-21) and mature sequences are shown in SEQ ID NOs: 70 and 71,respectively. The amino acid sequences for exemplary mouse LgR5precursor (with signal sequence, amino acids 1-21) and mature sequencesare shown in SEQ ID NOs: 72 and 73, respectively.

The term “LgR5-positive cancer” refers to a cancer comprising cells thatexpress LgR5 on their surface. For the purposes of determining whether acell expresses LgR5 on the surface, LgR5 mRNA expression is consideredto correlate to LgR5 expression on the cell surface. In someembodiments, expression of LgR5 mRNA is determined by a method selectedfrom in situ hybridization and RT-PCR (including quantitative RT-PCR).Alternatively, expression of LgR5 on the cell surface can be determined,for example, using antibodies to LgR5 in a method such asimmunohistochemistry, FACS, etc.

The term “LgR5-positive cell” refers to a cell that expresses LgR5 onits surface.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

“Alkyl” is C₁-C₁₈ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Examples are methyl (Me, —CH₃), ethyl (Et,—CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃.

The term “C₁-C₈ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 8 carbonatoms. Representative “C₁-C₈ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl,-n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while branched C₁-C₈ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C₁-C₈ alkylsinclude, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl,-isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl,-2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl,-acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl,-2-pentynyl, -3-methyl-1 butynyl. A C₁-C₈ alkyl group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where eachR′ is independently selected from H, —C₁-C₈ alkyl and aryl.

The term “C₁-C₁₂ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 12carbon atoms. A C₁-C₁₂ alkyl group can be unsubstituted or substitutedwith one or more groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

The term “C₁-C₆ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 6 carbonatoms. Representative “C₁-C₆ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -and n-hexyl; whilebranched C₁-C₆ alkyls include, but are not limited to, -isopropyl,-sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl;unsaturated C₁-C₆ alkyls include, but are not limited to, -vinyl,-allyl, -1-butenyl, -2-butenyl, and -isobutylenyl, -1-pentenyl,-2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl,-2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, and 3-hexyl. A C₁-C₆ alkylgroup can be unsubstituted or substituted with one or more groups, asdescribed above for C₁-C₈ alkyl group.

The term “C₁-C₄ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 4 carbonatoms. Representative “C₁-C₄ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl; while branched C₁-C₄ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl; unsaturated C₁-C₄ alkyls include, but are not limited to,-vinyl, -allyl, -1-butenyl, -2-butenyl, and -isobutylenyl. A C₁-C₄ alkylgroup can be unsubstituted or substituted with one or more groups, asdescribed above for C₁-C₈ alkyl group.

“Alkoxy” is an alkyl group singly bonded to an oxygen. Exemplary alkoxygroups include, but are not limited to, methoxy (—OCH₃) and ethoxy(—OCH₂CH₃). A “C₁-C₅ alkoxy” is an alkoxy group with 1 to 5 carbonatoms. Alkoxy groups may can be unsubstituted or substituted with one ormore groups, as described above for alkyl groups.

“Alkenyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond. Examples include, but are not limitedto: ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl(—C₅H₇), and 5-hexenyl (—CH₂CH₂CH₂CH₂CH═CH₂). A “C₂-C₈ alkenyl” is ahydrocarbon containing 2 to 8 normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond.

“Alkynyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond. Examples include, but are not limited to:acetylenic (—C≡CH) and propargyl (—CH₂C≡CH). A “C₂-C₈ alkynyl” is ahydrocarbon containing 2 to 8 normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond.

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—) 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

A “C₁-C₁₀ alkylene” is a straight chain, saturated hydrocarbon group ofthe formula —(CH₂)₁₋₁₀—. Examples of a C₁-C₁₀ alkylene includemethylene, ethylene, propylene, butylene, pentylene, hexylene,heptylene, ocytylene, nonylene and decalene.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to: 1,2-ethylene(—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to: acetylene (—C≡C—),propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡C—).

“Aryl” refers to a carbocyclic aromatic group. Examples of aryl groupsinclude, but are not limited to, phenyl, naphthyl and anthracenyl. Acarbocyclic aromatic group or a heterocyclic aromatic group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′,—OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₅-C₂₀ aryl” is an aryl group with 5 to 20 carbon atoms in thecarbocyclic aromatic rings. Examples of C₅-C₂₀ aryl groups include, butare not limited to, phenyl, naphthyl and anthracenyl. A C₅-C₂₀ arylgroup can be substituted or unsubstituted as described above for arylgroups. A “C₅-C₁₄ aryl” is an aryl group with 5 to 14 carbon atoms inthe carbocyclic aromatic rings. Examples of C₅-C₁₄ aryl groups include,but are not limited to, phenyl, naphthyl and anthracenyl. A C₅-C₁₄ arylgroup can be substituted or unsubstituted as described above for arylgroups.

An “arylene” is an aryl group which has two covalent bonds and can be inthe ortho, meta, or para configurations as shown in the followingstructures:

in which the phenyl group can be unsubstituted or substituted with up tofour groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. The arylalkyl group comprises 6 to 20 carbon atoms, e.g. the alkylmoiety, including alkanyl, alkenyl or alkynyl groups, of the arylalkylgroup is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbonatoms.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Typicalheteroarylalkyl groups include, but are not limited to,2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkylgroup comprises 6 to 20 carbon atoms, e.g. the alkyl moiety, includingalkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms and 1to 3 heteroatoms selected from N, O, P, and S. The heteroaryl moiety ofthe heteroarylalkyl group may be a monocycle having 3 to 7 ring members(2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), forexample: a bicyclo [4,5], [5,5], [5,6], or [6,6] system.

“Substituted alkyl,” “substituted aryl,” and “substituted arylalkyl”mean alkyl, aryl, and arylalkyl respectively, in which one or morehydrogen atoms are each independently replaced with a substituent.Typical substituents include, but are not limited to, —X, —R, —O⁻, —OR,—SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO,—NO₂, ═N₂, —N₃, NC(═O)R, —C(═O)R, —C(═O)NR₂, —SO₃ ⁻, —SO₃H, —S(═O)₂R,—OS(═O)₂OR, —S(═O)₂NR, —S(═O)R, —OP(═O)(OR)₂, —P(O)(OR)₂, —PO₃, —PO₃,H₂, —C(═O)R, —C(═O)X, —C(═S)R, —CO₂R, —CO₂, —C(═S)OR, —C(═O)SR,—C(═S)SR, —C(═O)NR₂, —C(═S)NR₂, —C(═NR)NR₂, where each X isindependently a halogen: F, Cl, Br, or I; and each R is independently—H, C₂-C₁₈ alkyl, C₆-C₂₀ aryl, C₃-C₁₄ heterocycle, protecting group orprodrug moiety. Alkylene, alkenylene, and alkynylene groups as describedabove may also be similarly substituted.

“Heteroaryl” and “heterocycle” refer to a ring system in which one ormore ring atoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 3 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6]system.

Exemplary heterocycles are described, e.g., in Paquette, Leo A.,“Principles of Modern Heterocyclic Chemistry” (W. A. Benjamin, New York,1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry ofHeterocyclic Compounds, A series of Monographs” (John Wiley & Sons, NewYork, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28;and J. Am. Chem. Soc. (1960) 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Still more typically, nitrogen bondedheterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl,1-pyrazolyl, and 1-piperidinyl.

A “C₃-C₈ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. Representative examples of a C₃-C₈ heterocycle include, but arenot limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl. A C₃-C₈ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₈ heterocyclo” refers to a C₃-C₈ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond. A C₃-C₈ heterocyclo can be unsubstituted or substituted with up tosix groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

A “C₃-C₂₀ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. A C₃-C₂₀ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₂₀ heterocyclo” refers to a C₃-C₂₀ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond.

“Carbocycle” means a saturated or unsaturated ring having 3 to 7 carbonatoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Monocycliccarbocycles have 3 to 6 ring atoms, still more typically 5 or 6 ringatoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g. arranged as abicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicyclo [5,6] or [6,6] system. Examples of monocycliccarbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl,and cyclooctyl.

A “C₃-C₈ carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated orunsaturated non-aromatic carbocyclic ring. Representative C₃-C₈carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl,-cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl,-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and-cyclooctadienyl. A C₃-C₈ carbocycle group can be unsubstituted orsubstituted with one or more groups including, but not limited to,—C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₃-C₈ carbocyclo” refers to a C₃-C₈ carbocycle group defined abovewherein one of the carbocycle groups' hydrogen atoms is replaced with abond.

“Linker” refers to a chemical moiety comprising a covalent bond or achain of atoms that covalently attaches an antibody to a drug moiety. Invarious embodiments, linkers include a divalent radical such as analkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as:—(CR₂)_(n)O(CR₂)_(n)—, repeating units of alkyloxy (e.g. polyethylenoxy,PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino,Jeffamine™); and diacid ester and amides including succinate,succinamide, diglycolate, malonate, and caproamide. In variousembodiments, linkers can comprise one or more amino acid residues, suchas valine, phenylalanine, lysine, and homolysine.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and 1 or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or 1 meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Certain leaving groups are well known in theart, and examples include, but are not limited to, a halide (e.g.,chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl(tosyl), trifluoromethylsulfonyl (triflate), andtrifluoromethylsulfonate.

The term “protecting group” refers to a substituent that is commonlyemployed to block or protect a particular functionality while reactingother functional groups on the compound. For example, an“amino-protecting group” is a substituent attached to an amino groupthat blocks or protects the amino functionality in the compound.Suitable amino-protecting groups include, but are not limited to,acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ)and 9-fluorenylmethylenoxycarbonyl (Fmoc). For a general description ofprotecting groups and their use, see T. W. Greene, Protective Groups inOrganic Synthesis, John Wiley & Sons, New York, 1991, or a lateredition.

II. Compositions and Methods

In one aspect, the invention is based, in part, on antibodies that bindto LgR5 and immunoconjugates comprising such antibodies. Antibodies andimmunoconjugates of the invention are useful, e.g., for the diagnosis ortreatment of LgR5-positive cancers.

A. Exemplary Anti-LgR5 Antibodies

In some embodiments, the invention provides isolated antibodies thatbind to LgR5. LgR5 is a seven-transmembrane protein found, for example,on the surface of actively cycling intestinal stem cells. Asdemonstrated herein, LgR5 is expressed in about 77% of colon tumorsections examined.

An exemplary naturally occurring human LgR5 precursor protein sequence,with signal sequence (amino acids 1-21) is provided in SEQ ID NO: 67,and the corresponding mature LgR5 protein sequence is shown in SEQ IDNO: 68 (corresponding to amino acids 22-907 of SEQ ID NO: 67).

In certain embodiments, an anti-LgR5 antibody has at least one or moreof the following characteristics, in any combination: (a) binds to anepitope within amino acids 22-555 of SEQ ID NO: 67; (b) binds LgR5 withan affinity of ≦5 nM, or ≦4 nM, or ≦3 nM, or ≦2 nM, or ≦1 nM, andoptionally ≧0.0001 nM, or ≧0.001 nM, or ≧0.01 nM; (c) does notsignificantly disrupt the binding of R-spondin (RSPO) to LgR5; (d) doesnot significantly disrupt beta-catenin signaling; (e) does notsignificantly disrupt RSPO activation of LgR5 signaling; (f) activatescaspase 3 cleavage; (g) recognizes both human and rodent LgR5; (h)recognizes human LgR5 but not rodent LgR5; (i) does not significantlyinhibit tumor growth in its unconjugated form; and (j) does not inducestem cell differentiation. In some embodiments, the anti-LgR5 antibodyis 8E11 and humanized variants thereof, such as hu8E11.v2; YW353; 2H6;and 3G12. In some embodiments, LgR5 is human LgR5. In some embodiments,LgR5 is selected from human, cynomolgus monkey, mouse, and rat LgR5.

(a) Binds to an Epitope within Amino Acids 22-555 of SEQ ID NO: 67

Methods of determining whether an anti-LgR5 antibody binds to an epitopeof LgR5 are known in the art. In some embodiments, binding of ananti-LgR5 antibody to an epitope of LgR5 (e.g., within amino acids22-555 of SEQ ID NO: 67) may be determined by expressing LgR5polypeptides with N- and C-terminal deletions in 293 cells and testingby FACS as described in Example I binding of the antibody to thetruncated polypeptides. In some embodiments, a substantial reduction(≧70% reduction) or elimination of binding of the antibody to atruncated polypeptide relative to binding to full-length LgR5 expressedin 293 cells indicates that the antibody does not bind to that truncatedpolypeptide. In some embodiments, LgR5 is human LgR5. In someembodiments, LgR5 is human LgR5 or cynomolgus monkey LgR5.

In some embodiments, the epitope of LgR5 comprises the lucine richN-terminal domain of LgR5 (e.g., amino acid residues 25-66 of SEQ IDNO:67). In some embodiments, the epitope of LgR5 comprises one or morelucine rich repeats (LRR) of LgR5 (e.g., amino acid residues 67-446 ofSEQ ID NO:67; LRRs 1-16 of LgR5).). In some embodiments, the epitope ofLgR5 comprises LRR 1 of LgR5 (e.g., amino acid residues 67-90 of SEQ IDNO:67). In some embodiments, the epitope of LgR5 comprises LRR 2 of LgR5(e g, amino acid residues 91-112 of SEQ ID NO:67). In some embodiments,the epitope of LgR5 comprises LRR 3 of LgR5 (e.g., amino acid residues115-136 of SEQ ID NO:67). In some embodiments, the epitope of LgR5comprises LRR 4 of LgR5 (e.g., amino acid residues 139-160 of SEQ IDNO:67). In some embodiments, the epitope of LgR5 comprises LRR 5 of LgR5(e.g., amino acid residues 163-184 of SEQ ID NO:67). In someembodiments, the epitope of LgR5 comprises LRR 6 of LgR5 (e.g., aminoacid residues 187-208 of SEQ ID NO:67). In some embodiments, the epitopeof LgR5 comprises LRR 7 of LgR5 (e.g., amino acid residues 211-232 ofSEQ ID NO:67). In some embodiments, the epitope of LgR5 comprises LRR 8of LgR5 (e.g., amino acid residues 235-256 of SEQ ID NO:67). In someembodiments, the epitope of LgR5 comprises LRR 9 of LgR5 (e.g., aminoacid residues 258-279 of SEQ ID NO:67). In some embodiments, the epitopeof LgR5 comprises LRR 10 of LgR5 (e.g., amino acid residues 282-303 ofSEQ ID NO:67). In some embodiments, the epitope of LgR5 comprises LRR 11of LgR5 (e.g., amino acid residues 306-328 of SEQ ID NO:67). In someembodiments, the epitope of LgR5 comprises LRR 12 of LgR5 (e.g., aminoacid residues 329-350 of SEQ ID NO:67). In some embodiments, the epitopeof LgR5 comprises LRR 13 of LgR5 (e.g., amino acid residues 353-374 ofSEQ ID NO:67). In some embodiments, the epitope of LgR5 comprises LRR 14of LgR5 (e.g., amino acid residues 375-396 of SEQ ID NO:67). In someembodiments, the epitope of LgR5 comprises LRR 15 of LgR5 (e.g., aminoacid residues 399-420 of SEQ ID NO:67). In some embodiments, the epitopeof LgR5 comprises LRR 16 of LgR5 (e.g., amino acid residues 423-446 ofSEQ ID NO:67). In some embodiments, the epitope of LgR5 comprises any ofLRR1 to LRR11, LRR2 to LRR11, LRR3 to LRR11, LLR1 to LLR3, LLR2 to LLR3,LLR2 to LLR8, LLR3 to LL7, or LLR4 to LLR6.

In some embodiments, the epitope of LgR5 comprises an epitope withinamino acids 22-555 of SEQ ID NO: 67. In some embodiments, the epitope ofLgR5 comprises an epitope within amino acids 22-424 of SEQ ID NO: 67. Insome embodiments, the epitope of LgR5 comprises an epitope within aminoacids 22-123 of SEQ ID NO: 67. In some embodiments, the epitope of LgR5comprises an epitope within amino acids 22-323 of SEQ ID NO: 67. In someembodiments, the epitope of LgR5 comprises an epitope within amino acids324-555 of SEQ ID NO: 67. In some embodiments, the epitope of LgR5comprises an epitope within amino acids 324-424 of SEQ ID NO: 67.

It is understood that aspect and embodiments described herein include“consisting” and/or “consisting effectively of” aspects and embodiments.

(b) Binds LgR5 with an Affinity of ≦5 nM, or ≦4 nM, or ≦3 nM, or ≦2 nM,or ≦1 nM, and Optionally ≧0.0001 nM, or ≧0.001 nM, or ≧0.01 nM

Methods of determining binding affinity are known in the art. In someembodiments, the binding affinity may be determined according to aBIAcore® assay as described herein in Example E. Specifically, in someembodiments, Kd may be measured using surface plasmon resonance assaysusing a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.). BIAcore™research grade CM5 chips may be activated with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) andN-hydroxysuccinimide (NHS) reagents according to the supplier'sinstructions. Goat anti-human Fc IgGs may be coupled to the chips toachieve approximately 10,000 response units (RU) in each flow cell.Unreacted coupling groups may be blocked with 1M ethanolamine. Forkinetics measurements, anti-LGR5 antibodies may be captured to achieveapproximately 300 RU. Two-fold serial dilutions of human LgR5 ECD (forexample, amino acids 22-557 (or a similar fragment, such as 22-555)fused to His-Fc expressed in a baculovirus system, or amino acids 22-558(or a similar fragment, such as 22-555) fused to Fc expressed from CHOcells; 125 nM to 0.49 nM) may be injected in HBS-P buffer (0.01M HEPESpH7.4, 0.15M NaCl, 0.005% surfactant P20) at 25° C. with a flow rate of30 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))may be calculated using a 1:1 Langmuir binding model (BIAcore™Evaluation Software version 3.2). The equilibrium dissociation constant(Kd) may be calculated as the ratio k_(off)/k_(on). If the on-rateexceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay above, thenthe on-rate may be determined by using a fluorescent quenching techniquethat measures the increase or decrease in fluorescence emissionintensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25°C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in thepresence of increasing concentrations of antigen as measured in aspectrometer, such as a stop-flow equipped spectrophotometer (AvivInstruments) or a 8000-series SLM-Aminco® spectrophotometer(ThermoSpectronic) with a stirred cuvette.

In some embodiments, the anti-LgR5 antibody binds LgR5 with an affinityof about any of ≦5 nM, or ≦4 nM, or ≦3 nM, or ≦2 nM, or ≦1 nM. In someembodiments, the anti-LgR5 antibody binds LgR5 with an affinity of about≦5. In some embodiments, the anti-LgR5 antibody binds LgR5 with anaffinity of about ≦4 nM. In some embodiments, the anti-LgR5 antibodybinds LgR5 with an affinity of about ≦3 nM. In some embodiments, theanti-LgR5 antibody binds LgR5 with an affinity of about ≦2 nM. In someembodiments, LgR5 is human LgR5. In some embodiments, LgR5 is human LgR5or cynomolgus monkey LgR5.

As is understood by one skilled in the art, reference to “about” a valueor parameter includes (and describes) embodiments that are direct tothat value or parameter per se. For example, description referring to“about X” includes description of “X”.

(c) Does not Significantly Disrupt the Binding of R-Spondin (RSPO) toLgR5

Methods of determining the ability of an anti-LgR5 antibody to disruptthe binding of an RSPO to LgR5 are known in the art. In someembodiments, the ability of an anti-LgR5 antibody to significantlydisrupt the binding of an R-spondon (RSPO) to LgR5 may be determined byflow cytometry. In some embodiments, for example, 293 cells expressingLgR5 may be contacted with fluorescently-labeled RSPO, such as RSPO1,RSPO2, RSPO3, and/or RSPO4, in the presence and absence of an anti-LgR5antibody. Binding of RSPO to the 293 cells may be detected usingfluorescence-activated cell sorting (FACS). In some embodiments, adecrease in RSPO binding in the presence of an anti-LgR5 antibody ofless than about 25% relative to RSPO binding in the presence of acontrol antibody, indicates that the anti-LgR5 antibody does notsignificantly disrupt binding of RSPO to LgR5.

In some embodiments, the ability of an anti-LgR5 antibody tosignificantly disrupt the binding of an R-spondon (RSPO) to LgR5 may bedetermined by BIAcore assay. In some embodiments, for example, LgR5extracellular domain may be immobilized on CM5 chips, e.g., as describedherein, and binding of RSPO, such as RSPO1, RSPO2, RSPO3, and/or RSPO4,to the immobilized LgR5 may be determined in the presence and absence ofan anti-LgR5 antibody. In some embodiments, a decrease in RSPO bindingin the presence of an anti-LgR5 antibody of less than about 25% relativeto RSPO binding in the presence of a control antibody, indicates thatthe anti-LgR5 antibody does not significantly disrupt binding of RSPO toLgR5.

In some embodiments, the RSPO is selected from RSPO1, RSPO2, RSPO3, andRSPO4. In some embodiments, the antibody disrupts binding by less thanabout 25%, less than about 20%, less than about 15%, or less than about10%. In some embodiments, the antibody does not detectably disruptbinding of an RSPO to LgR5. In some embodiments, LgR5 is human LgR5. Insome embodiments, LgR5 is human LgR5 or cynomolgus monkey LgR5.

(d) Does not Significantly Disrupt Wnt/Beta-Catenin Signaling

Methods of determining ability of an anti-LgR5 antibody to disruptwnt/beta-catenin signaling are known in the art. In some embodiments,the ability of an anti-LgR5 antibody to significantly disruptwnt/beta-catenin signaling may be determined using a reporter geneassay. In some embodiments, for example, a reporter construct comprisinga reporter gene (such as, for example, a luciferase gene) under thecontrol of a wnt/beta-catenin responsive promoter (such as, for example,a promoter comprising multimerized TCF/LEF DNA-binding sites) may betransfected into cells that express LgR5. The cells are then contactedwith a Wnt ligand, such as Wnt3a, and an RSPO, such as RSPO1, RSPO2,RSPO3, and/or RSPO4, in the presence and absence of an anti-LgR5antibody, and luciferase expression is measured. In some embodiments, adecrease in luciferase expression in the presence of antibody of lessthan about 25% relative to luciferase expression in the presence of acontrol antibody, indicates that the anti-LgR5 antibody does notsignificantly disrupt beta-catenin signaling.

In some embodiments, the antibody disrupts beta-catenin signaling byless than about 25%, less than about 20%, less than about 15%, or lessthan about 10%. In some embodiments, the antibody does not detectablydisrupt beta-catenin signaling. In some embodiments, LgR5 is human LgR5.In some embodiments, LgR5 is human LgR5 or cynomolgus monkey LgR5.

(e) does not Significantly Disrupt RSPO Activation of LgR5 Signaling

Methods of determining ability of an anti-LgR5 antibody to disrupt RSPOactivation of LgR5 are known in the art. In some embodiments, theability of an anti-LgR5 antibody to significantly disrupt RSPOactivation of LgR5 signaling may be determined using a reporter geneassay. In some embodiments, for example, a reporter construct comprisinga reporter gene (such as, for example, a luciferase gene) under thecontrol of a beta-catenin responsive promoter (such as, for example, apromoter comprising multimerized TCF/LEF DNA-binding sites) may betransfected into cells that express LgR5. The cells may be thencontacted with a Wnt ligand, such as Wnt3a, in the presence and absenceof an RSPO, such as RSPO1, RSPO2, RSPO3, and/or RSPO4, and theactivation of LgR5 signaling may be measured as the increase inluciferase expression in the presence of the RSPO. The activation ofLgR5 signaling may also be measured in the presence and absence of ananti-LgR5 antibody. In some embodiments, a decrease in the activation ofLgR5 signaling in the presence of RSPO1, RSPO2, RSPO3, and/or RSPO4 ofless than about 25% when the cells are contacted with an anti-LgR5antibody versus a control antibody, indicates that the anti-LgR5antibody does not significantly disrupt RSPO activation of LgR5signaling.

In some embodiments, the RSPO is selected from RSPO1, RSPO2, RSPO3, andRSPO4. In some embodiments, the antibody disrupts RSPO activation ofLgR5 signaling by less than about 25%, less than about 20%, less thanabout 15%, or less than about 10%. In some embodiments, the antibodydoes not detectably disrupt RSPO activation of LgR5 signaling. In someembodiments, LgR5 is human LgR5. In some embodiments, LgR5 is human LgR5or cynomolgus monkey LgR5.

(f) Activates Caspase 3 Cleavage

Methods of determining ability of an anti-LgR5 antibody to activatecaspase 3 cleavage are known in the art. In some embodiments, theability of an anti-LgR5 antibody to activate caspase 3 cleavage may bedetermined in a rodent xenograft model, e.g., as described in Example N.In some embodiments, the presence of cleaved caspase 3 may be measuredas a function of tumor area, for example, in formalin fixed paraffinembedded (FFPE) small intestine and colon tissue collected fromintestinal tumorogenesis model mice that were administered an anti-LgR5antibody. The presence of cleaved caspase 3 may be determined, in someembodiments, using immunohistochemistry. Further, in some embodiments,caspase 3 cleavage may be determined as a percent positive tumor area,e.g., as shown in Example N and FIG. 18.

In some embodiments, an anti-LgR5 antibody increases the percentage ofcaspase 3 positive tumor area according to the assay described inExample N by about any of at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 100% (i.e., thepercentage of positive tumor area doubles).

(g) Recognizes Both Human and Rodent LgR5

Methods of determining the ability of an anti-LgR5 antibody to bindhuman and rodent LgR5 are known in the art. In some embodiments, humanand rodent LgR5 polypeptides are expressed in 293 cells and binding ofthe antibody to the LgR5-expressing 293 cells is tested by FACS asdescribed in Example G. In some embodiments, rodent LgR5 is mouse or ratLgR5. In some embodiments, rodent LgR5 is mouse LgR5.

(h) Recognizes Human LgR5 but not Rodent LgR5

Methods of determining the ability of an anti-LgR5 antibody to bindhuman but not rodent LgR5 are known in the art. In some embodiments,human and rodent LgR5 polypeptides are expressed in 293 cells andbinding of the antibody to the LgR5-expressing 293 cells is tested byFACS as described in Example G. In some embodiments, rodent LgR5 ismouse or rat LgR5. In some embodiments, rodent LgR5 is mouse LgR5.

(i) Does not Significantly Inhibit Tumor Growth in its Unconjugated Form

Methods of determining the ability of an anti-LgR5 antibody to inhibittumor growth in its unconjugated form are known in the art. In someembodiments, a rodent xenograft model such as the D5124 pancreaticcancer xenograft model described in Example M is used. In someembodiments, an anti-LgR5 antibody does not significantly inhibit tumorgrowth in its unconjugated form in a LoVo colon cancer cell linexenograft model, for example, as described in Example L. In someembodiments, an anti-LgR5 antibody does not significantly inhibit tumorgrowth in its unconjugated form in a murine intestinal tumorigenesismodel, for example, as described in Example N. Inhibition of tumorgrowth in a xenograft model or murine intestinal tumorigenesis model isdetermined relative to a vehicle control or control antibody.

In some embodiments, an anti-LgR5 antibody inhibits tumor growth in itsunconjugated form by less than about 25%, less than about 20%, less thanabout 15%, or less than about 10%. In some embodiments, an anti-LgR5antibody does not detectably inhibit tumor growth in its unconjugatedform.

(j) Does not Induce Stem Cell Differentiation

Methods of determining the ability of an anti-LgR5 antibody to inducestem cell differentiation are known in the art. In some embodiments,stem cell differentiation may be assayed by determining ability todifferentiation of crypt base columnar cells (CBCs), which arefast-cycling stem cells in the small intestine that express LgR5, into,for example, enterocytes, goblet cells, and/or enteroendocrine cells, inthe presence and absence of an anti-LgR5 antibody In some embodiments,an anti-LgR5 antibody is considered to not induce stem celldifferentiation if about any of less than 25%, less than 20%, less than15%, or less than 10% of a population of CBCs differentiates in thepresence of the anti-LgR5 antibody under conditions in which a controlantibody also induces stem cell differentiation in less than about 25%of a population of CBCs.

In some embodiments, an anti-LgR5 antibody immunoconjugate inhibitstumor growth through a primary mechanism that is not inducing stem celldifferentiation. In some such embodiments, the anti-LgR5 antibodyimmunoconjugate inhibits tumor growth through cytotoxic activitymediated through a cytotoxic agent conjugated to the antibody in theimmunoconjugate.

Antibody 8E11 and Other Embodiments

In some embodiments, the invention provides an anti-LgR5 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 30; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 31; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 32; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 27; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 28; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 29.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 30; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 31; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 32. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO: 32. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO: 32 and HVR-L3comprising the amino acid sequence of SEQ ID NO: 29. In a furtherembodiment, the antibody comprises HVR-H3 comprising the amino acidsequence of SEQ ID NO: 32, HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 29, and HVR-H2 comprising the amino acid sequence of SEQ IDNO: 31. In a further embodiment, the antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 30; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 31; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 32.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 27; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 28; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 29. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO: 27; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO: 28; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:29.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 30, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 31, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO: 32; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 27, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 28, and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 29.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 30; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 31; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 32; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 27; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 28; and (0 HVR-L3comprising the amino acid sequence of SEQ ID NO: 29.

In any of the above embodiments, an anti-LgR5 antibody is humanized. Inone embodiment, an anti-LgR5 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaIV consensus (VL_(KIV)) framework and/or the VH framework VH₁. Incertain embodiments, the human acceptor framework is the human VL kappaIV consensus (VL_(KIV)) framework and/or the VH framework VH₁ comprisingan R71S mutation and an A78V mutation in heavy chain framework regionFR3.

In some embodiments, an anti-LgR5 antibody comprises HVRs as in any ofthe above embodiments, and further comprises a heavy chain framework FR3sequence selected from SEQ ID NOs: 40 to 43. In some embodiments, ananti-LgR5 antibody comprises HVRs as in any of the above embodiments,and further comprises a heavy chain framework FR3 sequence of SEQ ID NO:41. In some such embodiments, the heavy chain variable domain frameworkis a modified human VH₁ framework having an FR3 sequence selected fromSEQ ID NOs: 40 to 43. In some such embodiments, the heavy chain variabledomain framework is a modified human VH₁ framework having an FR3sequence of SEQ ID NO: 41.

In some embodiments, an anti-LgR5 antibody comprises HVRs as in any ofthe above embodiments, and further comprises a light chain framework FR3sequence of SEQ ID NO: 36. In some such embodiments, the heavy chainvariable domain framework is a modified VL kappa IV consensus (VL_(KIV))framework having an FR3 sequence of SEQ ID NO: 36.

In another aspect, an anti-LgR5 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, and 20. Incertain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequenceselected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, and 20 containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-LgR5 antibodycomprising that sequence retains the ability to bind to LgR5. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in a sequence selected from SEQ ID NOs: 6, 8,10, 12, 14, 16, 18, and 20. In certain embodiments, a total of 1 to 5amino acids have been substituted, inserted and/or deleted in a sequenceselected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, and 20. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the HVRs (i.e., in the FRs).

In some embodiments, an anti-LgR5 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 6. In some embodiments, an anti-LgR5 antibodycomprises a heavy chain variable domain (VH) sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 8. In someembodiments, an anti-LgR5 antibody comprises a heavy chain variabledomain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO: 10. In some embodiments, an anti-LgR5 antibody comprises aheavy chain variable domain (VH) sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO: 12. In some embodiments, an anti-LgR5antibody comprises a heavy chain variable domain (VH) sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 14. In someembodiments, an anti-LgR5 antibody comprises a heavy chain variabledomain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO: 16. In some embodiments, an anti-LgR5 antibody comprises aheavy chain variable domain (VH) sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO: 18. In some embodiments, an anti-LgR5antibody comprises a heavy chain variable domain (VH) sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 20.

Optionally, the anti-LgR5 antibody comprises the VH sequence selectedfrom SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, and 20, includingpost-translational modifications of that sequence. In a particularembodiment, the VH comprises one, two or three HVRs selected from: (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 30, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 31, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 32.

In another aspect, an anti-LgR5 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to an amino acid sequence selected from SEQ ID NOs: 5, 7, 9,11, 13, 15, 17, and 19. In certain embodiments, a VL sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to anamino acid sequence selected from SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17,and 19 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-LgR5 antibody comprising that sequence retains the ability to bindto LgR5. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in an amino acid sequenceselected from SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, and 19. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in an amino acid sequence selected from SEQ IDNOs: 5, 7, 9, 11, 13, 15, 17, and 19. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs).

In some embodiments, an anti-LgR5 antibody comprises a light chainvariable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 5. In some embodiments, an anti-LgR5 antibodycomprises a light chain variable domain (VL) sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 7. In someembodiments, an anti-LgR5 antibody comprises a light chain variabledomain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO: 9. In some embodiments, an anti-LgR5 antibody comprises alight chain variable domain (VL) sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO: 11. In some embodiments, an anti-LgR5antibody comprises a light chain variable domain (VL) sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 13. In someembodiments, an anti-LgR5 antibody comprises a light chain variabledomain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO: 15. In some embodiments, an anti-LgR5 antibody comprises alight chain variable domain (VL) sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO: 17. In some embodiments, an anti-LgR5antibody comprises a light chain variable domain (VL) sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 19.

Optionally, the anti-LgR5 antibody comprises the VL sequence of an aminoacid sequence selected from SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, and 19,including post-translational modifications of that sequence. In aparticular embodiment, the VL comprises one, two or three HVRs selectedfrom (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 27; (b)HVR-L2 comprising the amino acid sequence of SEQ ID NO: 28; and (c)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29.

In another aspect, an anti-LgR5 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In one embodiment, theantibody comprises the VH and VL sequences in SEQ ID NO: 6 and SEQ IDNO: 5, respectively, including post-translational modifications of thosesequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO: 8 and SEQ ID NO: 7, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO: 10 and SEQID NO: 9, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO: 12 and SEQ ID NO: 11, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO: 14 and SEQID NO: 13, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO: 16 and SEQ ID NO: 15, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO: 18 and SEQID NO: 17, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO: 20 and SEQ ID NO: 19, respectively, includingpost-translational modifications of those sequences.

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-LgR5 antibody provided herein. For example,in certain embodiments, an antibody is provided that binds to the sameepitope as an anti-LgR5 antibody comprising a VH sequence of SEQ ID NO:8 and a VL sequence of SEQ ID NO: 7. In certain embodiments, an antibodyis provided that binds to an epitope of SEQ ID NO: 67 from, within, oroverlapping amino acids 22-323. In some embodiments, an antibody isprovided that binds to an epitope of SEQ ID NO: 68 from, within, oroverlapping amino acids 1-312.

In a further aspect of the invention, an anti-LgR5 antibody according toany of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In one embodiment, an anti-LgR5antibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody,or F(ab′)₂ fragment. In another embodiment, the antibody is asubstantially full length antibody, e.g., an IgG1 antibody or otherantibody class or isotype as defined herein.

In a further aspect, an anti-LgR5 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below.

Antibody YW353 and Other Embodiments

In one aspect, the invention provides an anti-LgR5 antibody comprisingat least one, two, three, four, five, or six HVRs selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 60; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 61; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 62; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 57; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 58; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 59.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 60; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 61; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 62. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO: 62. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO: 62, and HVR-L3comprising the amino acid sequence of SEQ ID NO: 59. In a furtherembodiment, the antibody comprises HVR-H3 comprising the amino acidsequence of SEQ ID NO: 62, HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 59, and HVR-H2 comprising the amino acid sequence of SEQ IDNO: 61. In a further embodiment, the antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 60; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 61; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:62.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 57; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 58; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 59. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO: 57; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO: 58; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:59.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 60, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 61, and (iii) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 62; and (b) a VL domain comprising at least one, at least two, orall three VL HVR sequences selected from (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 57, (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 58, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 59.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 60; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 61; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 62; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 57; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 58; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO: 59.

In any of the above embodiments, an anti-LgR5 antibody is a humanantibody.

In another aspect, an anti-LgR5 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 26. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe amino acid sequence of SEQ ID NO: 26 contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-LgR5 antibody comprising that sequenceretains the ability to bind to LgR5. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO: 26. In certain embodiments, a total of 1 to 5 amino acidshave been substituted, inserted and/or deleted in SEQ ID NO: 26. Incertain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-LgR5antibody comprises the VH sequence of SEQ ID NO: 26, includingpost-translational modifications of that sequence. In a particularembodiment, the VH comprises one, two or three HVRs selected from: (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 60, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 61, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 62.

In another aspect, an anti-LgR5 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 25. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:25 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-LgR5 antibody comprising that sequence retains the ability to bindto LgR5. In certain embodiments, a total of 1 to 5 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO: 25. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 25. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-LgR5 antibody comprisesthe VL sequence of SEQ ID NO: 25, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 57; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 58; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 59.

In another aspect, an anti-LgR5 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In one embodiment, theantibody comprises the VH and VL sequences in SEQ ID NO: 26 and SEQ IDNO: 25, respectively, including post-translational modifications ofthose sequences.

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-LgR5 antibody provided herein. For example,in certain embodiments, an antibody is provided that binds to the sameepitope as an anti-LgR5 antibody comprising a VH sequence of SEQ ID NO:26 and a VL sequence of SEQ ID NO: 25. In certain embodiments, anantibody is provided that binds to an epitope of SEQ ID NO: 67 from,within, or overlapping amino acids 22-123. In certain embodiments, anantibody is provided that binds to an epitope of SEQ ID NO: 68 from,within, or overlapping amino acids 1-102.

In a further aspect of the invention, an anti-LgR5 antibody according toany of the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-LgR5 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG2a antibody or other antibody class or isotype asdefined herein.

In a further aspect, an anti-LgR5 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below.

Antibody 3G12 and Other Embodiments

In some embodiments, the invention provides an anti-LgR5 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 48; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 50; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 45; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 46; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 47.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 48; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 49; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 50. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO: 50. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO: 50 and HVR-L3comprising the amino acid sequence of SEQ ID NO: 47. In a furtherembodiment, the antibody comprises HVR-H3 comprising the amino acidsequence of SEQ ID NO: 50, HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 47, and HVR-H2 comprising the amino acid sequence of SEQ IDNO: 49. In a further embodiment, the antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 48; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 49; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 50.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 45; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 46; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 47. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO: 45; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO: 46; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:47.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 48, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 49, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO: 50; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 45, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 46, and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 47.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 48; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 49; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 50; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 45; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 46; and (0 HVR-L3comprising the amino acid sequence of SEQ ID NO: 47.

In any of the above embodiments, an anti-LgR5 antibody is humanized. Inone embodiment, an anti-LgR5 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaconsensus (VL_(K)) framework and/or the human VH subgroup 3 consensus(VH₃) framework.

In another aspect, an anti-LgR5 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 22. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe amino acid sequence of SEQ ID NO: 22 contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-LgR5 antibody comprising that sequenceretains the ability to bind to LgR5. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inthe amino acid sequence of SEQ ID NO: 22. In certain embodiments, atotal of 1 to 5 amino acids have been substituted, inserted and/ordeleted in the amino acid sequence of SEQ ID NO: 22. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the HVRs (i.e., in the FRs).

Optionally, the anti-LgR5 antibody comprises the VH sequence of SEQ IDNO: 22, including post-translational modifications of that sequence. Ina particular embodiment, the VH comprises one, two or three HVRsselected from: (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 48, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49,and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50.

In another aspect, an anti-LgR5 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 21. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:21 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-LgR5 antibody comprising that sequence retains the ability to bindto LgR5. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in the amino acid sequence ofSEQ ID NO: 21. In certain embodiments, a total of 1 to 5 amino acidshave been substituted, inserted and/or deleted in the amino acidsequence of SEQ ID NO: 21. In certain embodiments, the substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs).

Optionally, the anti-LgR5 antibody comprises the VL sequence of SEQ IDNO: 21, including post-translational modifications of that sequence. Ina particular embodiment, the VL comprises one, two or three HVRsselected from (a) HVR-L1 comprising the amino acid sequence of SEQ IDNO: 45; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 46;and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 47.

In another aspect, an anti-LgR5 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In one embodiment, theantibody comprises the VH and VL sequences in SEQ ID NO: 22 and SEQ IDNO: 21, respectively, including post-translational modifications ofthose sequences.

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-LgR5 antibody provided herein. For example,in certain embodiments, an antibody is provided that binds to the sameepitope as an anti-LgR5 antibody comprising a VH sequence of SEQ ID NO:22 and a VL sequence of SEQ ID NO: 21. In certain embodiments, anantibody is provided that binds to an epitope of SEQ ID NO: 67 from,within, or overlapping amino acids 324-423. In some embodiments, anantibody is provided that binds to an epitope of SEQ ID NO: 68 from,within, or overlapping amino acids 303-402. In certain embodiments, anantibody is provided that binds to an epitope of SEQ ID NO: 67 from,within, or overlapping amino acids 324-555. In some embodiments, anantibody is provided that binds to an epitope of SEQ ID NO: 68 from,within, or overlapping amino acids 303-534.

In a further aspect of the invention, an anti-LgR5 antibody according toany of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In one embodiment, an anti-LgR5antibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody,or F(ab′)₂ fragment. In another embodiment, the antibody is asubstantially full length antibody, e.g., an IgG1 antibody or otherantibody class or isotype as defined herein.

In a further aspect, an anti-LgR5 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below.

Antibody 2H6 and Other Embodiments

In some embodiments, the invention provides an anti-LgR5 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 55; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 56; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 51; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 52; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 53.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 55; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 56. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO: 56. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO: 56 and HVR-L3comprising the amino acid sequence of SEQ ID NO: 53. In a furtherembodiment, the antibody comprises HVR-H3 comprising the amino acidsequence of SEQ ID NO: 56, HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 53, and HVR-H2 comprising the amino acid sequence of SEQ IDNO: 55. In a further embodiment, the antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 54; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 55; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 56.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 52; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 53. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO: 51; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO: 52; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:53.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 54, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 55, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO: 56; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 51, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 52, and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 53.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 55; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 56; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 51; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 52; and (0 HVR-L3comprising the amino acid sequence of SEQ ID NO: 53.

In any of the above embodiments, an anti-LgR5 antibody is humanized. Inone embodiment, an anti-LgR5 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaconsensus (VL_(K)) framework and/or the human VH subgroup 3 (VH₃)framework.

In another aspect, an anti-LgR5 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 24. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe amino acid sequence of SEQ ID NO: 24 contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-LgR5 antibody comprising that sequenceretains the ability to bind to LgR5. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inthe amino acid sequence of SEQ ID NO: 24. In certain embodiments, atotal of 1 to 5 amino acids have been substituted, inserted and/ordeleted in the amino acid sequence of SEQ ID NO: 24. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the HVRs (i.e., in the FRs).

Optionally, the anti-LgR5 antibody comprises the VH sequence of SEQ IDNO: 24, including post-translational modifications of that sequence. Ina particular embodiment, the VH comprises one, two or three HVRsselected from: (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 54, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 55,and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56.

In another aspect, an anti-LgR5 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 23. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:23 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-LgR5 antibody comprising that sequence retains the ability to bindto LgR5. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in the amino acid sequence ofSEQ ID NO: 23. In certain embodiments, a total of 1 to 5 amino acidshave been substituted, inserted and/or deleted in the amino acidsequence of SEQ ID NO: 23. In certain embodiments, the substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs).

Optionally, the anti-LgR5 antibody comprises the VL sequence of theamino acid sequence of SEQ ID NO: 23, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 51; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 52; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 53.

In another aspect, an anti-LgR5 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In one embodiment, theantibody comprises the VH and VL sequences in SEQ ID NO: 24 and SEQ IDNO: 23, respectively, including post-translational modifications ofthose sequences.

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-LgR5 antibody provided herein. For example,in certain embodiments, an antibody is provided that binds to the sameepitope as an anti-LgR5 antibody comprising a VH sequence of SEQ ID NO:24 and a VL sequence of SEQ ID NO: 23. In certain embodiments, anantibody is provided that binds to an epitope of SEQ ID NO: 67 from,within, or overlapping amino acids 324-423. In some embodiments, anantibody is provided that binds to an epitope of SEQ ID NO: 68 from,within, or overlapping amino acids 303-402. In certain embodiments, anantibody is provided that binds to an epitope of SEQ ID NO: 67 from,within, or overlapping amino acids 324-555. In some embodiments, anantibody is provided that binds to an epitope of SEQ ID NO: 68 from,within, or overlapping amino acids 303-534.

In a further aspect of the invention, an anti-LgR5 antibody according toany of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In one embodiment, an anti-LgR5antibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody,or F(ab′)₂ fragment. In another embodiment, the antibody is asubstantially full length antibody, e.g., an IgG1 antibody or otherantibody class or isotype as defined herein.

In a further aspect, an anti-LgR5 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below.

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or≦0.001 nM, and optionally is ≧10⁻¹³ M. (e.g. 10⁻⁸M or less, e.g. from10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881 (1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 μM or 26 μM antigen are mixedwith serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HuMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbori. J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for LgR5 and the other is for any other antigen. Incertain embodiments, one of the binding specificities is for LgR5 andthe other is for CD3. See, e.g., U.S. Pat. No. 5,821,337. In certainembodiments, bispecific antibodies may bind to two different epitopes ofLgR5. Bispecific antibodies may also be used to localize cytotoxicagents to cells which express LgR5. Bispecific antibodies can beprepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S.Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tuft et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to LgR5 as well asanother, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: H is, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex is usedto identify contact points between the antibody and antigen. Suchcontact residues and neighboring residues may be targeted or eliminatedas candidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e.g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and 5400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

An exemplary hu8E11.v2 light chain (LC) V205C thiomab has the heavychain and light chain sequences of SEQ ID NOs: 64 and 74, respectively.An exemplary hu8E11.v2 heavy chain (HC) A118C thiomab has the heavychain and light chain sequences of SEQ ID NOs: 75 and 63, respectively.An exemplary hu8E11.v2 heavy chain (HC) S400C thiomab has the heavychain and light chain sequences of SEQ ID NOs: 76 and 63, respectively.

An exemplary YW353 light chain (LC) V205C thiomab has the heavy chainand light chain sequences of SEQ ID NOs: 66 and 77, respectively. Anexemplary YW353 heavy chain (HC) A118C thiomab has the heavy chain andlight chain sequences of SEQ ID NOs: 78 and 65, respectively. Anexemplary YW353 heavy chain (HC) S400C thiomab has the heavy chain andlight chain sequences of SEQ ID NOs: 79 and 65, respectively.

Further exemplary V205C cysteine engineered thiomabs comprise a lightchain comprising a variable region selected from SEQ ID NOs: 3, 5, 7, 9,11, 13, 15, 17, 19, 21, and 23 and a constant region of SEQ ID NO: 80;and a heavy chain comprising a variable region selected from SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 and a human heavy chainconstant region, such as an IgG1. Further exemplary A118C cysteineengineered thiomabs comprise a light chain comprising a variable regionselected from SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 anda human light chain constant region, such as a kappa light chainconstant region; and a heavy chain comprising a variable region selectedfrom SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 and aconstant region of SEQ ID NO: 81. Further exemplary S400C cysteineengineered thiomabs comprise a light chain comprising a variable regionselected from SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 anda human light chain constant region, such as a kappa light chainconstant region; and a heavy chain comprising a variable region selectedfrom SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 and aconstant region of SEQ ID NO: 82.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethyleneglycol propionaldehyde may have advantages in manufacturing due to itsstability in water. The polymer may be of any molecular weight, and maybe branched or unbranched. The number of polymers attached to theantibody may vary, and if more than one polymer are attached, they canbe the same or different molecules. In general, the number and/or typeof polymers used for derivatization can be determined based onconsiderations including, but not limited to, the particular propertiesor functions of the antibody to be improved, whether the antibodyderivative will be used in a therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-LgR5 antibody described herein isprovided. Such nucleic acid may encode an amino acid sequence comprisingthe VL and/or an amino acid sequence comprising the VH of the antibody(e.g., the light and/or heavy chains of the antibody). In a furtherembodiment, one or more vectors (e.g., expression vectors) comprisingsuch nucleic acid are provided. In a further embodiment, a host cellcomprising such nucleic acid is provided. In one such embodiment, a hostcell comprises (e.g., has been transformed with): (1) a vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and an amino acid sequence comprising the VH ofthe antibody, or (2) a first vector comprising a nucleic acid thatencodes an amino acid sequence comprising the VL of the antibody and asecond vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-LgR5 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-LgR5 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TR1 cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-LgR5 antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, BIACore®, FACS,or Western blot.

In another aspect, competition assays may be used to identify anantibody that competes with any of the antibodies described herein forbinding to LgR5. In certain embodiments, such a competing antibody bindsto the same epitope (e.g., a linear or a conformational epitope) that isbound by an antibody described herein. Detailed exemplary methods formapping an epitope to which an antibody binds are provided in Morris(1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol.66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized LgR5 is incubated in asolution comprising a first labeled antibody that binds to LgR5 (e.g.,any of the antibodies described herein) and a second unlabeled antibodythat is being tested for its ability to compete with the first antibodyfor binding to LgR5. The second antibody may be present in a hybridomasupernatant. As a control, immobilized LgR5 is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to LgR5, excess unbound antibody is removed, and theamount of label associated with immobilized LgR5 is measured. If theamount of label associated with immobilized LgR5 is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to LgR5. See Harlow and Lane (1988) Antibodies: A LaboratoryManual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-LgR5antibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes(i.e., a radioconjugate).

Immunoconjugates allow for the targeted delivery of a drug moiety to atumor, and, in some embodiments intracellular accumulation therein,where systemic administration of unconjugated drugs may result inunacceptable levels of toxicity to normal cells (Polakis P. (2005)Current Opinion in Pharmacology 5:382-387).

Antibody-drug conjugates (ADC) are targeted chemotherapeutic moleculeswhich combine properties of both antibodies and cytotoxic drugs bytargeting potent cytotoxic drugs to antigen-expressing tumor cells(Teicher, B. A. (2009) Current Cancer Drug Targets 9:982-1004), therebyenhancing the therapeutic index by maximizing efficacy and minimizingoff-target toxicity (Carter, P. J. and Senter P. D. (2008) The CancerJour. 14(3):154-169; Chari, R. V. (2008) Acc. Chem. Res. 41:98-107.

The ADC compounds of the invention include those with anticanceractivity. In some embodiments, the ADC compounds include an antibodyconjugated, i.e. covalently attached, to the drug moiety. In someembodiments, the antibody is covalently attached to the drug moietythrough a linker. The antibody-drug conjugates (ADC) of the inventionselectively deliver an effective dose of a drug to tumor tissue wherebygreater selectivity, i.e. a lower efficacious dose, may be achievedwhile increasing the therapeutic index (“therapeutic window”).

The drug moiety (D) of the antibody-drug conjugates (ADC) may includeany compound, moiety or group that has a cytotoxic or cytostatic effect.Drug moieties may impart their cytotoxic and cytostatic effects bymechanisms including but not limited to tubulin binding, DNA binding orintercalation, and inhibition of RNA polymerase, protein synthesis,and/or topoisomerase. Exemplary drug moieties include, but are notlimited to, a maytansinoid, dolastatin, auristatin, calicheamicin,pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives,PNU-159682, anthracycline, duocarmycin, vinca alkaloid, taxane,trichothecene, CC1065, camptothecin, elinafide, and stereoisomers,isosteres, analogs, and derivatives thereof that have cytotoxicactivity. Nonlimiting examples of such immunoconjugates are discussed infurther detail below.

1. Exemplary Antibody-Drug Conjugates

An exemplary embodiment of an antibody-drug conjugate (ADC) compoundcomprises an antibody (Ab) which targets a tumor cell, a drug moiety(D), and a linker moiety (L) that attaches Ab to D. In some embodiments,the antibody is attached to the linker moiety (L) through one or moreamino acid residues, such as lysine and/or cysteine.

An exemplary ADC has Formula I:Ab-(L-D)_(p)  Iwhere p is 1 to about 20. In some embodiments, the number of drugmoieties that can be conjugated to an antibody is limited by the numberof free cysteine residues. In some embodiments, free cysteine residuesare introduced into the antibody amino acid sequence by the methodsdescribed herein. Exemplary ADC of Formula I include, but are notlimited to, antibodies that have 1, 2, 3, or 4 engineered cysteine aminoacids (Lyon, R. et al (2012) Methods in Enzym. 502:123-138). In someembodiments, one or more free cysteine residues are already present inan antibody, without the use of engineering, in which case the existingfree cysteine residues may be used to conjugate the antibody to a drug.In some embodiments, an antibody is exposed to reducing conditions priorto conjugation of the antibody in order to generate one or more freecysteine residues.

a) Exemplary Linkers

A “Linker” (L) is a bifunctional or multifunctional moiety that can beused to link one or more drug moieties (D) to an antibody (Ab) to forman antibody-drug conjugate (ADC) of Formula I. In some embodiments,antibody-drug conjugates (ADC) can be prepared using a Linker havingreactive functionalities for covalently attaching to the drug and to theantibody. For example, in some embodiments, a cysteine thiol of anantibody (Ab) can form a bond with a reactive functional group of alinker or a drug-linker intermediate to make an ADC.

In one aspect, a linker has a functionality that is capable of reactingwith a free cysteine present on an antibody to form a covalent bond.Nonlimiting exemplary such reactive functionalities include maleimide,haloacetamides, α-haloacetyl, activated esters such as succinimideesters, 4-nitrophenyl esters, pentafluorophenyl esters,tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates, and isothiocyanates. See, e.g., the conjugationmethod at page 766 of Klussman, et al (2004), Bioconjugate Chemistry15(4):765-773, and the Examples herein.

In some embodiments, a linker has a functionality that is capable ofreacting with an electrophilic group present on an antibody. Exemplarysuch electrophilic groups include, but are not limited to, aldehyde andketone carbonyl groups. In some embodiments, a heteroatom of thereactive functionality of the linker can react with an electrophilicgroup on an antibody and form a covalent bond to an antibody unit.Nonlimiting exemplary such reactive functionalities include, but are notlimited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide.

A linker may comprise one or more linker components. Exemplary linkercomponents include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”),valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), and4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“MCC”). Various linkercomponents are known in the art, some of which are described below.

A linker may be a “cleavable linker,” facilitating release of a drug.Nonlimiting exemplary cleavable linkers include acid-labile linkers(e.g., comprising hydrazone), protease-sensitive (e.g.,peptidase-sensitive) linkers, photolabile linkers, ordisulfide-containing linkers (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020).

In certain embodiments, a linker has the following Formula II:-A_(a)-W_(w)—Y_(y)—  II

wherein A is a “stretcher unit”, and a is an integer from 0 to 1; W isan “amino acid unit”, and w is an integer from 0 to 12; Y is a “spacerunit”, and y is 0, 1, or 2. An ADC comprising the linker of Formula IIhas the Formula I(A): Ab-(A_(a)-W_(w)—Y_(y)-D)_(p), wherein Ab, D, and pare defined as above for Formula I. Exemplary embodiments of suchlinkers are described in U.S. Pat. No. 7,498,298, which is expresslyincorporated herein by reference.

In some embodiments, a linker component comprises a “stretcher unit” (A)that links an antibody to another linker component or to a drug moiety.Nonlimiting exemplary stretcher units are shown below (wherein the wavyline indicates sites of covalent attachment to an antibody, drug, oradditional linker components):

In some embodiments, a linker component comprises an “amino acid unit”(W). In some such embodiments, the amino acid unit allows for cleavageof the linker by a protease, thereby facilitating release of the drugfrom the immunoconjugate upon exposure to intracellular proteases, suchas lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol.21:778-784). Exemplary amino acid units include, but are not limited to,dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplarydipeptides include, but are not limited to, valine-citrulline (vc orval-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine(fk or phe-lys); phenylalanine-homolysine (phe-homolys); andN-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include,but are not limited to, glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). An amino acid unit may compriseamino acid residues that occur naturally and/or minor amino acids and/ornon-naturally occurring amino acid analogs, such as citrulline Aminoacid units can be designed and optimized for enzymatic cleavage by aparticular enzyme, for example, a tumor-associated protease, cathepsinB, C and D, or a plasmin protease.

Typically, peptide-type linkers can be prepared by forming a peptidebond between two or more amino acids and/or peptide fragments. Suchpeptide bonds can be prepared, for example, according to a liquid phasesynthesis method (e.g., E. Schroder and K. Lübke (1965) “The Peptides”,volume 1, pp 76-136, Academic Press).

In some embodiments, a linker component comprises a “spacer unit” (Y)that links the antibody to a drug moiety, either directly or through astretcher unit and/or an amino acid unit. A spacer unit may be“self-immolative” or a “non-self-immolative.” A “non-self-immolative”spacer unit is one in which part or all of the spacer unit remains boundto the drug moiety upon cleavage of the ADC. Examples ofnon-self-immolative spacer units include, but are not limited to, aglycine spacer unit and a glycine-glycine spacer unit. In someembodiments, enzymatic cleavage of an ADC containing a glycine-glycinespacer unit by a tumor-cell associated protease results in release of aglycine-glycine-drug moiety from the remainder of the ADC. In some suchembodiments, the glycine-glycine-drug moiety is subjected to ahydrolysis step in the tumor cell, thus cleaving the glycine-glycinespacer unit from the drug moiety.

A “self-immolative” spacer unit allows for release of the drug moiety.In certain embodiments, a spacer unit of a linker comprises ap-aminobenzyl unit. In some such embodiments, a p-aminobenzyl alcohol isattached to an amino acid unit via an amide bond, and a carbamate,methylcarbamate, or carbonate is made between the benzyl alcohol and thedrug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005)15:1087-1103). In some embodiments, the spacer unit comprisesp-aminobenzyloxycarbonyl (PAB). In some embodiments, an ADC comprising aself-immolative linker has the structure:

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro, or-cyano; m is an integer ranging from 0 to 4; X may be one or moreadditional spacer units or may be absent; and p ranges from 1 to about20. In some embodiments, p ranges from 1 to 10, 1 to 7, 1 to 5, or 1 to4. Nonlimiting exemplary X spacer units include:

wherein R₁ and R₂ are independently selected from H and C₁-C₆ alkyl. Insome embodiments, R₁ and R₂ are each —CH₃.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB group,such as 2-aminoimidazol-5-methanol derivatives (U.S. Pat. No. 7,375,078;Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- orpara-aminobenzylacetals. In some embodiments, spacers can be used thatundergo cyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides (Rodrigues et al (1995)Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (Storm et al (1972) J. Amer. Chem. Soc.94:5815) and 2-aminophenylpropionic acid amides (Amsberry, et al (1990)J. Org. Chem. 55:5867). Linkage of a drug to the α-carbon of a glycineresidue is another example of a self-immolative spacer that may beuseful in ADC (Kingsbury et al (1984) J. Med. Chem. 27:1447).

In some embodiments, linker L may be a dendritic type linker forcovalent attachment of more than one drug moiety to an antibody througha branching, multifunctional linker moiety (Sun et al (2002) Bioorganic& Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003) Bioorganic& Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase themolar ratio of drug to antibody, i.e. loading, which is related to thepotency of the ADC. Thus, where an antibody bears only one reactivecysteine thiol group, a multitude of drug moieties may be attachedthrough a dendritic linker.

Nonlimiting exemplary linkers are shown below in the context of an ADCof Formula I:

wherein R₁ and R₂ are independently selected from H and C₁-C₆ alkyl. Insome embodiments, R₁ and R₂ are each —CH₃.

wherein n is 0 to 12. In some embodiments, n is 2 to 10. In someembodiments, n is 4 to 8.

Further nonlimiting exemplary ADCs include the structures:

each R is independently H or C₁-C₆ alkyl; and n is 1 to 12.

In some embodiments, a linker is substituted with groups that modulatesolubility and/or reactivity. As a nonlimiting example, a chargedsubstituent such as sulfonate (—SO₃ ⁻) or ammonium may increase watersolubility of the linker reagent and facilitate the coupling reaction ofthe linker reagent with the antibody and/or the drug moiety, orfacilitate the coupling reaction of Ab-L (antibody-linker intermediate)with D, or D-L (drug-linker intermediate) with Ab, depending on thesynthetic route employed to prepare the ADC. In some embodiments, aportion of the linker is coupled to the antibody and a portion of thelinker is coupled to the drug, and then the Ab-(linker portion)^(a) iscoupled to drug-(linker portion)^(b) to form the ADC of Formula I.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with the following linker reagents:bis-maleimido-trioxyethylene glycol (BMPEO),N-(β-maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS),N-(ε-maleimidocaproyloxy) succinimide ester (EMCS),N-[γ-maleimidobutyryloxy]succinimide ester (GMBS),1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),4-(4-N-Maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA),succinimidyl (4-iodoacetyl)aminobenzoate (SIAB),N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl6-[(beta-maleimidopropionamido)hexanoate] (SMPH), iminothiolane (IT),sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and succinimidyl-(4-vinylsulfone)benzoate (SVSB), andincluding bis-maleimide reagents: dithiobismaleimidoethane (DTME),1,4-Bismaleimidobutane (BMB), 1,4 Bismaleimidyl-2,3-dihydroxybutane(BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)₂(shown below), and BM(PEG)₃ (shown below); bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCl), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In someembodiments, bis-maleimide reagents allow the attachment of the thiolgroup of a cysteine in the antibody to a thiol-containing drug moiety,linker, or linker-drug intermediate. Other functional groups that arereactive with thiol groups include, but are not limited to,iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyldisulfide, isocyanate, and isothiocyanate.

Certain useful linker reagents can be obtained from various commercialsources, such as Pierce Biotechnology, Inc. (Rockford, Ill.), MolecularBiosciences Inc. (Boulder, Colo.), or synthesized in accordance withprocedures described in the art; for example, in Toki et al (2002) J.Org. Chem. 67:1866-1872; Dubowchik, et al. (1997) Tetrahedron Letters,38:5257-60; Walker, M. A. (1995) J. Org. Chem. 60:5352-5355; Frisch etal (1996) Bioconjugate Chem. 7:180-186; U.S. Pat. No. 6,214,345; WO02/088172; US2003130189; US2003096743; WO 03/026577; WO 03/043583; andWO 04/032828.

Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, e.g., WO94/11026.

b) Exemplary Drug Moieties

(1) Maytansine and Maytansinoids

In some embodiments, an immunoconjugate comprises an antibody conjugatedto one or more maytansinoid molecules. Maytansinoids are derivatives ofmaytansine, and are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinoids are disclosed, for example, in U.S. Pat. Nos.4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757;4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929;4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219;4,450,254; 4,362,663; and 4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody-drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification or derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through non-disulfide linkers to antibodies,(iii) stable in plasma, and (iv) effective against a variety of tumorcell lines.

Certain maytansinoids suitable for use as maytansinoid drug moieties areknown in the art and can be isolated from natural sources according toknown methods or produced using genetic engineering techniques (see,e.g., Yu et al (2002) PNAS 99:7968-7973). Maytansinoids may also beprepared synthetically according to known methods.

Exemplary maytansinoid drug moieties include, but are not limited to,those having a modified aromatic ring, such as: C-19-dechloro (U.S. Pat.No. 4,256,746) (prepared, for example, by lithium aluminum hydridereduction of ansamytocin P2); C-20-hydroxy (orC-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016)(prepared, for example, by demethylation using Streptomyces orActinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared,for example, by acylation using acyl chlorides), and those havingmodifications at other positions of the aromatic ring.

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared, forexample, by the reaction of maytansinol with H₂S or P₂S₅);C-14-alkoxymethyl(demethoxy/CH₂OR)(U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared, for example, from Nocardia); C-15-hydroxy/acyloxy(U.S. Pat. No. 4,364,866) (prepared, for example, by the conversion ofmaytansinol by Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and4,315,929) (for example, isolated from Trewia nudlflora);C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared, forexample, by the demethylation of maytansinol by Streptomyces); and4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared, for example, by thetitanium trichloride/LAH reduction of maytansinol).

Many positions on maytansinoid compounds are useful as the linkageposition. For example, an ester linkage may be formed by reaction with ahydroxyl group using conventional coupling techniques. In someembodiments, the reaction may occur at the C-3 position having ahydroxyl group, the C-14 position modified with hydroxymethyl, the C-15position modified with a hydroxyl group, and the C-20 position having ahydroxyl group. In some embodiments, the linkage is formed at the C-3position of maytansinol or a maytansinol analogue.

Maytansinoid drug moieties include those having the structure:

where the wavy line indicates the covalent attachment of the sulfur atomof the maytansinoid drug moiety to a linker of an ADC. Each R mayindependently be H or a C₁-C₆ alkyl. The alkylene chain attaching theamide group to the sulfur atom may be methanyl, ethanyl, or propyl,i.e., m is 1, 2, or 3 (U.S. Pat. No. 633,410; U.S. Pat. No. 5,208,020;Chari et al (1992) Cancer Res. 52:127-131; Liu et al (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623).

All stereoisomers of the maytansinoid drug moiety are contemplated forthe ADC of the invention, i.e. any combination of R and S configurationsat the chiral carbons (U.S. Pat. No. 7,276,497; U.S. Pat. No. 6,913,748;U.S. Pat. No. 6,441,163; U.S. Pat. No. 633,410 (RE39151); U.S. Pat. No.5,208,020; Widdison et al (2006) J. Med. Chem. 49:4392-4408, which areincorporated by reference in their entirety). In some embodiments, themaytansinoid drug moiety has the following stereochemistry:

Exemplary embodiments of maytansinoid drug moieties include, but are notlimited to, DM1; DM3; and DM4, having the structures:

wherein the wavy line indicates the covalent attachment of the sulfuratom of the drug to a linker (L) of an antibody-drug conjugate.

Other exemplary maytansinoid antibody-drug conjugates have the followingstructures and abbreviations (wherein Ab is antibody and p is 1 to about20. In some embodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is1 to 4):

Exemplary antibody-drug conjugates where DM1 is linked through a BMPEOlinker to a thiol group of the antibody have the structure andabbreviation:

where Ab is antibody; n is 0, 1, or 2; and p is 1 to about 20. In someembodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is 1 to 4.

Immunoconjugates containing maytansinoids, methods of making the same,and their therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020 and 5,416,064; US 2005/0276812 A1; and European Patent EP 0425 235 B1, the disclosures of which are hereby expressly incorporatedby reference. See also Liu et al. Proc. Natl. Acad. Sci. USA93:8618-8623 (1996); and Chari et al. Cancer Research 52:127-131 (1992).

In some embodiments, antibody-maytansinoid conjugates may be prepared bychemically linking an antibody to a maytansinoid molecule withoutsignificantly diminishing the biological activity of either the antibodyor the maytansinoid molecule. See, e.g., U.S. Pat. No. 5,208,020 (thedisclosure of which is hereby expressly incorporated by reference). Insome embodiments, ADC with an average of 3-4 maytansinoid moleculesconjugated per antibody molecule has shown efficacy in enhancingcytotoxicity of target cells without negatively affecting the functionor solubility of the antibody. In some instances, even one molecule oftoxin/antibody is expected to enhance cytotoxicity over the use of nakedantibody.

Exemplary linking groups for making antibody-maytansinoid conjugatesinclude, for example, those described herein and those disclosed in U.S.Pat. No. 5,208,020; EP Patent 0 425 235 B1; Chari et al. Cancer Research52:127-131 (1992); US 2005/0276812 A1; and US 2005/016993 A1, thedisclosures of which are hereby expressly incorporated by reference.

(2) Auristatins and Dolastatins

Drug moieties include dolastatins, auristatins, and analogs andderivatives thereof (U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588;U.S. Pat. No. 5,767,237; U.S. Pat. No. 6,124,431). Auristatins arederivatives of the marine mollusk compound dolastatin-10. While notintending to be bound by any particular theory, dolastatins andauristatins have been shown to interfere with microtubule dynamics, GTPhydrolysis, and nuclear and cellular division (Woyke et al (2001)Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer(U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998)Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin/auristatindrug moiety may be attached to the antibody through the N (amino)terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO02/088172; Doronina et al (2003) Nature Biotechnology 21(7):778-784;Francisco et al (2003) Blood 102(4):1458-1465).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties D_(E) and D_(F), disclosed in U.S.Pat. No. 7,498,298 and U.S. Pat. No. 7,659,241, the disclosures of whichare expressly incorporated by reference in their entirety:

wherein the wavy line of D_(E) and D_(F) indicates the covalentattachment site to an antibody or antibody-linker component, andindependently at each location:

R² is selected from H and C₁-C₈ alkyl;

R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selectedfrom H, C₁-C₈ alkyl and C₃-C₈ carbocycle and n is selected from 2, 3, 4,5 and 6;

R⁶ is selected from H and C₁-C₈ alkyl;

R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈carbocycle and O—(C₁-C₈ alkyl);

R⁹ is selected from H and C₁-C₈ alkyl;

R¹⁰ is selected from aryl or C₃-C₈ heterocycle;

Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;

R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,—(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₈ alkyl;

R¹⁴ is H or C₁-C₈ alkyl;

each occurrence of e is independently H, COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈ alkyl;

each occurrence of e is independently H, C₁-C₈ alkyl, or—(CH₂)_(n)—COOH;

R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); and

n is an integer ranging from 0 to 6.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H or methyl. In an exemplary embodiment, R³ and R⁴are each isopropyl, R⁵ is —H, and R⁷ is sec-butyl.

In yet another embodiment, R² and R⁶ are each methyl, and R⁹ is —H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment, R¹⁰ is aryl.

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is —O—, R¹¹ is —H, methyl or t-butyl.

In one embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—N(R¹⁶)₂, and R¹⁶ is —C₁-C₈ alkyl or —(CH₂)_(n)—COOH.

In another embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—SO₃H.

An exemplary auristatin embodiment of formula D_(E) is MMAE, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

An exemplary auristatin embodiment of formula D_(E) is MMAF, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

Other exemplary embodiments include monomethylvaline compounds havingphenylalanine carboxy modifications at the C-terminus of thepentapeptide auristatin drug moiety (WO 2007/008848) andmonomethylvaline compounds having phenylalanine sidechain modificationsat the C-terminus of the pentapeptide auristatin drug moiety (WO2007/008603).

Nonlimiting exemplary embodiments of ADC of Formula I comprising MMAE orMMAF and various linker components have the following structures andabbreviations (wherein “Ab” is an antibody; p is 1 to about 8, “Val-Cit”is a valine-citrulline dipeptide; and “S” is a sulfur atom:

Nonlimiting exemplary embodiments of ADCs of Formula I comprising MMAFand various linker components further include Ab-MC-PAB-MMAF andAb-PAB-MMAF. Immunoconjugates comprising MMAF attached to an antibody bya linker that is not proteolytically cleavable have been shown topossess activity comparable to immunoconjugates comprising MMAF attachedto an antibody by a proteolytically cleavable linker (Doronina et al.(2006) Bioconjugate Chem. 17:114-124). In some such embodiments, drugrelease is believed to be effected by antibody degradation in the cell.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to a liquidphase synthesis method (see, e.g., E. Schröder and K. Lübke, “ThePeptides”, volume 1, pp 76-136, 1965, Academic Press).Auristatin/dolastatin drug moieties may, in some embodiments, beprepared according to the methods of: U.S. Pat. No. 7,498,298; U.S. Pat.No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem.Soc. 111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; Pettit et al(1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat.Biotechnol. 21(7):778-784.

In some embodiments, auristatin/dolastatin drug moieties of formulasD_(E) such as MMAE, and D_(E), such as MMAF, and drug-linkerintermediates and derivatives thereof, such as MC-MMAF, MC-MMAE,MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE, may be prepared using methodsdescribed in U.S. Pat. No. 7,498,298; Doronina et al. (2006)Bioconjugate Chem. 17:114-124; and Doronina et al. (2003) Nat. Biotech.21:778-784 and then conjugated to an antibody of interest.

(3) Calicheamicin

In some embodiments, the immunoconjugate comprises an antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics, and analogues thereof, are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations (Hinman etal., (1993) Cancer Research 53:3336-3342; Lode et al., (1998) CancerResearch 58:2925-2928). Calicheamicin has intracellular sites of actionbut, in certain instances, does not readily cross the plasma membrane.Therefore, cellular uptake of these agents through antibody-mediatedinternalization may, in some embodiments, greatly enhances theircytotoxic effects. Nonlimiting exemplary methods of preparingantibody-drug conjugates with a calicheamicin drug moiety are described,for example, in U.S. Pat. No. 5,712,374; U.S. Pat. No. 5,714,586; U.S.Pat. No. 5,739,116; and U.S. Pat. No. 5,767,285.

(4) Pyrrolobenzodiazepines

In some embodiments, an ADC comprises a pyrrolobenzodiazepine (PBD). Insome embodiments, PDB dimers recognize and bind to specific DNAsequences. The natural product anthramycin, a PBD, was first reported in1965 (Leimgruber, et al., (1965)J. Am. Chem. Soc., 87:5793-5795;Leimgruber, et al., (1965)J. Am. Chem. Soc., 87:5791-5793). Since then,a number of PBDs, both naturally-occurring and analogues, have beenreported (Thurston, et al., (1994) Chem. Rev. 1994, 433-465 includingdimers of the tricyclic PBD scaffold (U.S. Pat. No. 6,884,799; U.S. Pat.No. 7,049,311; U.S. Pat. No. 7,067,511; U.S. Pat. No. 7,265,105; U.S.Pat. No. 7,511,032; U.S. Pat. No. 7,528,126; U.S. Pat. No. 7,557,099).Without intending to be bound by any particular theory, it is believedthat the dimer structure imparts the appropriate three-dimensional shapefor isohelicity with the minor groove of B-form DNA, leading to a snugfit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, NewYork, pp. 3-11 (1975); Hurley and Needham-VanDevanter, (1986) Acc. Chem.Res., 19:230-237). Dimeric PBD compounds bearing C2 aryl substituentshave been shown to be useful as cytotoxic agents (Hartley et al (2010)Cancer Res. 70(17):6849-6858; Antonow (2010) J. Med. Chem.53(7):2927-2941; Howard et al (2009) Bioorganic and Med. Chem. Letters19(22):6463-6466).

PBD dimers have been conjugated to antibodies and the resulting ADCshown to have anti-cancer properties. Nonlimiting exemplary linkagesites on the PBD dimer include the five-membered pyrrolo ring, thetether between the PBD units, and the N10-C11 imine group (WO2009/016516; US 2009/304710; US 2010/047257; US 2009/036431; US2011/0256157; WO 2011/130598).

Nonlimiting exemplary PBD dimer components of ADCs are of Formula A:

and salts and solvates thereof, wherein:

the wavy line indicates the covalent attachment site to the linker;

the dotted lines indicate the optional presence of a double bond betweenC1 and C2 or C2 and C3;

R² is independently selected from H, OH, ═O, ═CH₂, CN, R, OR, ═CH—R^(D),═C(R^(D))₂, O—SO₂—R, CO₂R and COR, and optionally further selected fromhalo or dihalo, wherein R^(D) is independently selected from R, CO₂R,COR, CHO, CO₂H, and halo;

R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,NHR, NRR′, NO₂, Me₃Sn and halo;

R⁷ is independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo;

Q is independently selected from O, S and NH;

R¹¹ is either H, or R or, where Q is O, SO₃M, where M is a metal cation;

R and R′ are each independently selected from optionally substitutedC₁₋₈ alkyl, C₁₋₁₂ alkyl, C₃₋₈ heterocyclyl, C₃₋₂₀ heterocycle, and C₅₋₂₀aryl groups, and optionally in relation to the group NRR′, R and R′together with the nitrogen atom to which they are attached form anoptionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring;

R¹², R¹⁶, R¹⁹ and R¹⁷ are as defined for R², R⁶, R⁹ and R⁷ respectively;

R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one ormore heteroatoms, e.g. O, S, N(H), NMe and/or aromatic rings, e.g.benzene or pyridine, which rings are optionally substituted; and

X and X′ are independently selected from O, S and N(H).

In some embodiments, R and R′ are each independently selected fromoptionally substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocycle, and C₅₋₂₀ arylgroups, and optionally in relation to the group NRR′, R and R′ togetherwith the nitrogen atom to which they are attached form an optionallysubstituted 4-, 5-, 6- or 7-membered heterocyclic ring.

In some embodiments, R⁹ and R¹⁹ are H.

In some embodiments, R⁶ and R¹⁶ are H.

In some embodiments, R⁷ are R¹⁷ are both OR^(7A), where R^(7A) isoptionally substituted C₁₋₄ alkyl. In some embodiments, R^(7A) is Me. Insome embodiments, R^(7A) is Ch₂Ph, where Ph is a phenyl group.

In some embodiments, X is O.

In some embodiments, R¹¹ is H.

In some embodiments, there is a double bond between C2 and C3 in eachmonomer unit.

In some embodiments, R² and R¹² are independently selected from H and R.In some embodiments, R² and R¹² are independently R. In someembodiments, R² and R¹² are independently optionally substituted C₅₋₂₀aryl or C₅₋₇ aryl or C₈₋₁₀ aryl. In some embodiments, R² and R¹² areindependently optionally substituted phenyl, thienyl, napthyl, pyridyl,quinolinyl, or isoquinolinyl. In some embodiments, R² and R¹² areindependently selected from ═O, ═CH₂, ═CH—R^(D), and ═C(R^(D))₂. In someembodiments, R² and R¹² each ═CH₂. In some embodiments, R² and R¹² areeach H. In some embodiments, R² and R¹² are each ═O. In someembodiments, R² and R¹² are each ═CF₂. In some embodiments, R² and/orR¹² are independently ═C(R^(D))₂. In some embodiments, R² and/or R¹² areindependently ═CH—R^(D).

In some embodiments, when R² and/or R¹² is ═CH—R^(D), each group mayindependently have either configuration shown below:

In some embodiments, a ═CH—R^(D) is in configuration (I).

In some embodiments, R″ is a C₃ alkylene group or a C₅ alkylene group.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(I):

wherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(II):

wherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(III):

wherein R^(E) and R^(E)″ are each independently selected from H orR^(D), wherein R^(D) is defined as above; andwherein n is 0 or 1.

In some embodiments, n is 0. In some embodiments, n is 1. In someembodiments, R^(E) and/or R^(E)″ is H. In some embodiments, R^(E) andR^(E)″ are H. In some embodiments, R^(E) and/or R^(E)″ is R^(D), whereinR^(D) is optionally substituted C₁₋₁₂ alkyl. In some embodiments, R^(E)and/or R^(E)″ is R^(D), wherein R^(D) is methyl.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(IV):

wherein Ar¹ and Ar² are each independently optionally substituted C₅₋₂₀aryl; wherein Ar¹ and Ar² may be the same or different; andwherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(V):

wherein Ar¹ and Ar² are each independently optionally substituted C₅₋₂₀aryl; wherein Ar¹ and Ar² may be the same or different; and

wherein n is 0 or 1.

In some embodiments, Ar¹ and Ar² are each independently selected fromoptionally substituted phenyl, furanyl, thiophenyl and pyridyl. In someembodiments, Ar¹ and Ar² are each independently optionally substitutedphenyl. In some embodiments, Ar¹ and Ar² are each independentlyoptionally substituted thien-2-yl or thien-3-yl. In some embodiments,Ar¹ and Ar² are each independently optionally substituted quinolinyl orisoquinolinyl. The quinolinyl or isoquinolinyl group may be bound to thePBD core through any available ring position. For example, thequinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4-yl,quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. In someembodiments, the quinolinyl is selected from quinolin-3-yl andquinolin-6-yl. The isoquinolinyl may be isoquinolin-1-yl,isoquinolin-3-yl, isoquinolin-4-yl, isoquinolin-5-yl, isoquinolin-6-yl,isoquinolin-7-yl and isoquinolin-8-yl. In some embodiments, theisoquinolinyl is selected from isoquinolin-3-yl and isoquinolin-6-yl.

Further nonlimiting exemplary PBD dimer components of ADCs are ofFormula B:

and salts and solvates thereof, wherein:

the wavy line indicates the covalent attachment site to the linker;

the wavy line connected to the OH indicates the S or R configuration;

R^(V1) and R^(V2) are independently selected from H, methyl, ethyl andphenyl (which phenyl may be optionally substituted with fluoro,particularly in the 4 position) and C₅₋₆ heterocyclyl; wherein R^(V1)and R^(V2) may be the same or different; and

n is 0 or 1.

In some embodiments, R^(V1) and R^(V2) are independently selected fromH, phenyl, and 4-fluorophenyl.

In some embodiments, a linker may be attached at one of various sites ofthe PBD dimer drug moiety, including the N10 imine of the B ring, theC-2 endo/exo position of the C ring, or the tether unit linking the Arings (see structures C(I) and C(II) below).

Nonlimiting exemplary PBD dimer components of ADCs include Formulas C(I)and C(II):

Formulas C(I) and C(II) are shown in their N10-C11 imine form. ExemplaryPBD drug moieties also include the carbinolamine and protectedcarbinolamine forms as well, as shown in the table below:

wherein:

X is CH₂ (n=1 to 5), N, or O;

Z and Z′ are independently selected from OR and NR₂, where R is aprimary, secondary or tertiary alkyl chain containing 1 to 5 carbonatoms;

R₁, R₁′, R₂ and R₂′ are each independently selected from H, C₁-C₈ alkyl,C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₅₋₂₀ aryl (including substituted aryls),C₅₋₂₀ heteroaryl groups, —NH₂, —NHMe, —OH, and —SH, where, in someembodiments, alkyl, alkenyl and alkynyl chains comprise up to 5 carbonatoms;

R₃ and R₃′ are independently selected from H, OR, NHR, and NR₂, where Ris a primary, secondary or tertiary alkyl chain containing 1 to 5 carbonatoms;

R₄ and R₄′ are independently selected from H, Me, and OMe;

R₅ is selected from C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₅₋₂₀aryl (including aryls substituted by halo, nitro, cyano, alkoxy, alkyl,heterocyclyl) and C₅₋₂₀ heteroaryl groups, where, in some embodiments,alkyl, alkenyl and alkynyl chains comprise up to 5 carbon atoms;

R₁₁ is H, C₁-C₈ alkyl, or a protecting group (such as acetyl,trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ),9-fluorenylmethylenoxycarbonyl (Fmoc), or a moiety comprising aself-immolating unit such as valine-citrulline-PAB);

R₁₂ is H, C₁-C₈ alkyl, or a protecting group;

wherein a hydrogen of one of R₁, R₁′, R₂, R₂′, R₅, or R₁₂ or a hydrogenof the —OCH₂CH₂(X)—CH₂CH₂O— spacer between the A rings is replaced witha bond connected to the linker of the ADC.

Exemplary PDB dimer portions of ADC include, but are not limited to (thewavy line indicates the site of covalent attachment to the linker):

Nonlimiting exemplary embodiments of ADCs comprising PBD dimers have thefollowing structures:

wherein:

n is 0 to 12. In some embodiments, n is 2 to 10. In some embodiments, nis 4 to 8. In some embodiments, n is selected from 4, 5, 6, 7, and 8.

The linkers of PBD dimer-val-cit-PAB-Ab and the PBD dimer-Phe-Lys-PAB-Abare protease cleavable, while the linker of PBD dimer-maleimide-acetalis acid-labile.

PBD dimers and ADC comprising PBD dimers may be prepared according tomethods known in the art. See, e.g., WO 2009/016516; US 2009/304710; US2010/047257; US 2009/036431; US 2011/0256157; WO 2011/130598.

(5) Anthracyclines

In some embodiments, an ADC comprising anthracycline. Anthracyclines areantibiotic compounds that exhibit cytotoxic activity. While notintending to be bound by any particular theory, studies have indicatedthat anthracyclines may operate to kill cells by a number of differentmechanisms, including: 1) intercalation of the drug molecules into theDNA of the cell thereby inhibiting DNA-dependent nucleic acid synthesis;2) production by the drug of free radicals which then react withcellular macromolecules to cause damage to the cells, and/or 3)interactions of the drug molecules with the cell membrane (see, e.g., C.Peterson et al., “Transport And Storage Of Anthracycline In ExperimentalSystems And Human Leukemia” in Anthracycline Antibiotics In CancerTherapy; N. R. Bachur, “Free Radical Damage” id. at pp. 97-102). Becauseof their cytotoxic potential anthracyclines have been used in thetreatment of numerous cancers such as leukemia, breast carcinoma, lungcarcinoma, ovarian adenocarcinoma and sarcomas (see e.g., P. H-Wiernik,in Anthracycline: Current Status And New Developments p 11).

Nonlimiting exemplary anthracyclines include doxorubicin, epirubicin,idarubicin, daunomycin, nemorubicin, and derivatives thereof.Immunoconjugates and prodrugs of daunorubicin and doxorubicin have beenprepared and studied (Kratz et al (2006) Current Med. Chem. 13:477-523;Jeffrey et al (2006) Bioorganic & Med. Chem. Letters 16:358-362; Torgovet al (2005) Bioconj. Chem. 16:717-721; Nagy et al (2000) Proc. Natl.Acad. Sci. USA 97:829-834; Dubowchik et al (2002) Bioorg. & Med. Chem.Letters 12:1529-1532; King et al (2002) J. Med. Chem. 45:4336-4343; EP0328147; U.S. Pat. No. 6,630,579). The antibody-drug conjugateBR96-doxorubicin reacts specifically with the tumor-associated antigenLewis-Y and has been evaluated in phase I and II studies (Saleh et al(2000) J. Clin. Oncology 18:2282-2292; Ajani et al (2000) Cancer Jour.6:78-81; Tolcher et al (1999) J. Clin. Oncology 17:478-484).

PNU-159682 is a potent metabolite (or derivative) of nemorubicin(Quintieri, et al. (2005) Clinical Cancer Research 11(4):1608-1617).Nemorubicin is a semisynthetic analog of doxorubicin with a2-methoxymorpholino group on the glycoside amino of doxorubicin and hasbeen under clinical evaluation (Grandi et al (1990) Cancer Treat. Rev.17:133; Ripamonti et al (1992) Brit. J. Cancer 65:703;), including phaseII/III trials for hepatocellular carcinoma (Sun et al (2003) Proceedingsof the American Society for Clinical Oncology 22, Abs1448; Quintieri(2003) Proceedings of the American Association of Cancer Research,44:1st Ed, Abs 4649; Pacciarini et al (2006) Jour. Clin. Oncology24:14116).

A nonlimiting exemplary ADC comprising nemorubicin or nemorubicinderivatives is shown in Formula Ia:

wherein R₁ is hydrogen atom, hydroxy or methoxy group and R₂ is a C₁-C₅alkoxy group, or a pharmaceutically acceptable salt thereof;

L₁ and Z together are a linker (L) as described herein;

T is an antibody (Ab) as described herein; and

m is 1 to about 20. In some embodiments, m is 1 to 10, 1 to 7, 1 to 5,or 1 to 4.

In some embodiments, R₁ and R₂ are both methoxy (—OMe).

A further nonlimiting exemplary ADC comprising nemorubicin ornemorubicin derivatives is shown in Formula Ib:

wherein R₁ is hydrogen atom, hydroxy or methoxy group and R₂ is a C₁-C₅alkoxy group, or a pharmaceutically acceptable salt thereof;

L₂ and Z together are a linker (L) as described herein;

T is an antibody (Ab) as described herein; and

m is 1 to about 20. In some embodiments, m is 1 to 10, 1 to 7, 1 to 5,or 1 to 4.

In some embodiments, R₁ and R₂ are both methoxy (—OMe).

In some embodiments, the nemorubicin component of anemorubicin-containing ADC is PNU-159682. In some such embodiments, thedrug portion of the ADC may have one of the following structures:

wherein the wavy line indicates the attachment to the linker (L).

Anthracyclines, including PNU-159682, may be conjugated to antibodiesthrough several linkage sites and a variety of linkers (US 2011/0076287;WO2009/099741; US 2010/0034837; WO 2010/009124), including the linkersdescribed herein.

Exemplary ADCs comprising a nemorubicin and linker include, but are notlimited to:

wherein:

R₁ and R₂ are independently selected from H and C₁-C₆ alkyl; and

The linker of PNU-159682 maleimide acetal-Ab is acid-labile, while thelinkers of PNU-159682-val-cit-PAB-Ab, PNU-159682-val-cit-PAB-spacer-Ab,and PNU-159682-val-cit-PAB-spacer(R¹R²)-Ab are protease cleavable.

(6) Other Drug Moieties

Drug moieties also include geldanamycin (Mandler et al (2000) J. Nat.Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791); and enzymatically active toxins and fragments thereof,including, but not limited to, diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.See, e.g., WO 93/21232.

Drug moieties also include compounds with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease).

In certain embodiments, an immunoconjugate may comprise a highlyradioactive atom. A variety of radioactive isotopes are available forthe production of radioconjugated antibodies. Examples include At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactiveisotopes of Lu. In some embodiments, when an immunoconjugate is used fordetection, it may comprise a radioactive atom for scintigraphic studies,for example Tc⁹⁹ or I¹²³, or a spin label for nuclear magnetic resonance(NMR) imaging (also known as magnetic resonance imaging, MRI), such aszirconium-89, iodine-123, iodine-131, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.Zirconium-89 may be complexed to various metal chelating agents andconjugated to antibodies, e.g., for PET imaging (WO 2011/056983).

The radio- or other labels may be incorporated in the immunoconjugate inknown ways. For example, a peptide may be biosynthesized or chemicallysynthesized using suitable amino acid precursors comprising, forexample, one or more fluorine-19 atoms in place of one or morehydrogens. In some embodiments, labels such as Tc⁹⁹, I¹²³, Re¹⁸⁶, Re¹⁸⁸and In¹¹¹ can be attached via a cysteine residue in the antibody. Insome embodiments, yttrium-90 can be attached via a lysine residue of theantibody. In some embodiments, the IODOGEN method (Fraker et al (1978)Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporateiodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRCPress 1989) describes certain other methods.

In certain embodiments, an immunoconjugate may comprise an antibodyconjugated to a prodrug-activating enzyme. In some such embodiments, aprodrug-activating enzyme converts a prodrug (e.g., a peptidylchemotherapeutic agent, see WO 81/01145) to an active drug, such as ananti-cancer drug. Such immunoconjugates are useful, in some embodiments,in antibody-dependent enzyme-mediated prodrug therapy (“ADEPT”). Enzymesthat may be conjugated to an antibody include, but are not limited to,alkaline phosphatases, which are useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatases, which areuseful for converting sulfate-containing prodrugs into free drugs;cytosine deaminase, which is useful for converting non-toxic5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases,such as serratia protease, thermolysis, subtilisin, carboxypeptidasesand cathepsins (such as cathepsins B and L), which are useful forconverting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, which are useful for converting prodrugs thatcontain D-amino acid substituents; carbohydrate-cleaving enzymes such asβ-galactosidase and neuraminidase, which are useful for convertingglycosylated prodrugs into free drugs; β-lactamase, which is useful forconverting drugs derivatized with β-lactams into free drugs; andpenicillin amidases, such as penicillin V amidase and penicillin Gamidase, which are useful for converting drugs derivatized at theiramine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,into free drugs. In some embodiments, enzymes may be covalently bound toantibodies by recombinant DNA techniques well known in the art. See,e.g., Neuberger et al., Nature 312:604-608 (1984).

c) Drug Loading

Drug loading is represented by p, the average number of drug moietiesper antibody in a molecule of Formula I. Drug loading may range from 1to 20 drug moieties (D) per antibody. ADCs of Formula I includecollections of antibodies conjugated with a range of drug moieties, from1 to 20. The average number of drug moieties per antibody inpreparations of ADC from conjugation reactions may be characterized byconventional means such as mass spectroscopy, ELISA assay, and HPLC. Thequantitative distribution of ADC in terms of p may also be determined.In some instances, separation, purification, and characterization ofhomogeneous ADC where p is a certain value from ADC with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in certain exemplary embodiments above, an antibodymay have only one or several cysteine thiol groups, or may have only oneor several sufficiently reactive thiol groups through which a linker maybe attached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the average drug loading for an ADC ranges from 1 to about8; from about 2 to about 6; or from about 3 to about 5. Indeed, it hasbeen shown that for certain ADCs, the optimal ratio of drug moieties perantibody may be less than 8, and may be about 2 to about 5 (U.S. Pat.No. 7,498,298).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, and for example, by: (i) limiting the molar excess ofdrug-linker intermediate or linker reagent relative to antibody, (ii)limiting the conjugation reaction time or temperature, and (iii) partialor limiting reductive conditions for cysteine thiol modification.

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent, then theresulting product is a mixture of ADC compounds with a distribution ofone or more drug moieties attached to an antibody. The average number ofdrugs per antibody may be calculated from the mixture by a dual ELISAantibody assay, which is specific for antibody and specific for thedrug. Individual ADC molecules may be identified in the mixture by massspectroscopy and separated by HPLC, e.g. hydrophobic interactionchromatography (see, e.g., McDonagh et al (2006) Prot. Engr. Design &Selection 19(7):299-307; Hamblett et al (2004) Clin. Cancer Res.10:7063-7070; Hamblett, K. J., et al. “Effect of drug loading on thepharmacology, pharmacokinetics, and toxicity of an anti-CD30antibody-drug conjugate,” Abstract No. 624, American Association forCancer Research, 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings ofthe AACR, Volume 45, March 2004; Alley, S. C., et al. “Controlling thelocation of drug attachment in antibody-drug conjugates,” Abstract No.627, American Association for Cancer Research, 2004 Annual Meeting, Mar.27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certainembodiments, a homogeneous ADC with a single loading value may beisolated from the conjugation mixture by electrophoresis orchromatography.

d) Certain Methods of Preparing Immunoconjugates

An ADC of Formula I may be prepared by several routes employing organicchemistry reactions, conditions, and reagents known to those skilled inthe art, including: (1) reaction of a nucleophilic group of an antibodywith a bivalent linker reagent to form Ab-L via a covalent bond,followed by reaction with a drug moiety D; and (2) reaction of anucleophilic group of a drug moiety with a bivalent linker reagent, toform D-L, via a covalent bond, followed by reaction with a nucleophilicgroup of an antibody. Exemplary methods for preparing an ADC of FormulaI via the latter route are described in U.S. Pat. No. 7,498,298, whichis expressly incorporated herein by reference.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; and (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol) or tricarbonylethylphosphine (TCEP), such that theantibody is fully or partially reduced. Each cysteine bridge will thusform, theoretically, two reactive thiol nucleophiles. Additionalnucleophilic groups can be introduced into antibodies throughmodification of lysine residues, e.g., by reacting lysine residues with2-iminothiolane (Traut's reagent), resulting in conversion of an amineinto a thiol. Reactive thiol groups may also be introduced into anantibody by introducing one, two, three, four, or more cysteine residues(e.g., by preparing variant antibodies comprising one or more non-nativecysteine amino acid residues).

Antibody-drug conjugates of the invention may also be produced byreaction between an electrophilic group on an antibody, such as analdehyde or ketone carbonyl group, with a nucleophilic group on a linkerreagent or drug. Useful nucleophilic groups on a linker reagent include,but are not limited to, hydrazide, oxime, amino, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In oneembodiment, an antibody is modified to introduce electrophilic moietiesthat are capable of reacting with nucleophilic substituents on thelinker reagent or drug. In another embodiment, the sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither galactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the antibody that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, antibodies containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Such analdehyde can be reacted with a drug moiety or linker nucleophile.

Exemplary nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Nonlimiting exemplary cross-linker reagents that may be used to prepareADC are described herein in the section titled “Exemplary Linkers.”Methods of using such cross-linker reagents to link two moieties,including a proteinaceous moiety and a chemical moiety, are known in theart. In some embodiments, a fusion protein comprising an antibody and acytotoxic agent may be made, e.g., by recombinant techniques or peptidesynthesis. A recombinant DNA molecule may comprise regions encoding theantibody and cytotoxic portions of the conjugate either adjacent to oneanother or separated by a region encoding a linker peptide which doesnot destroy the desired properties of the conjugate.

In yet another embodiment, an antibody may be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pre-targeting whereinthe antibody-receptor conjugate is administered to the patient, followedby removal of unbound conjugate from the circulation using a clearingagent and then administration of a “ligand” (e.g., avidin) which isconjugated to a cytotoxic agent (e.g., a drug or radionucleotide).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-LgR5 antibodies provided hereinis useful for detecting the presence of LgR5 in a biological sample. Theterm “detecting” as used herein encompasses quantitative or qualitativedetection. A “biological sample” comprises, e.g., a cell or tissue(e.g., biopsy material, including cancerous or potentially cancerouscolon, colorectal, small intestine, endometrial, pancreatic, or ovariantissue).

In one embodiment, an anti-LgR5 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of LgR5 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-LgR5 antibody as described herein under conditionspermissive for binding of the anti-LgR5 antibody to LgR5, and detectingwhether a complex is formed between the anti-LgR5 antibody and LgR5 inthe biological sample. Such method may be an in vitro or in vivo method.In one embodiment, an anti-LgR5 antibody is used to select subjectseligible for therapy with an anti-LgR5 antibody, e.g. where LgR5 is abiomarker for selection of patients. In a further embodiment, thebiological sample is a cell or tissue (e.g., biopsy material, includingcancerous or potentially cancerous colon, colorectal, small intestine,endometrial, pancreatic, or ovarian tissue).

In a further embodiment, an anti-LgR5 antibody is used in vivo todetect, e.g., by in vivo imaging, an LgR5-positive cancer in a subject,e.g., for the purposes of diagnosing, prognosing, or staging cancer,determining the appropriate course of therapy, or monitoring response ofa cancer to therapy. One method known in the art for in vivo detectionis immuno-positron emission tomography (immuno-PET), as described, e.g.,in van Dongen et al., The Oncologist 12:1379-1389 (2007) and Verel etal., J. Nucl. Med. 44:1271-1281 (2003). In such embodiments, a method isprovided for detecting an LgR5-positive cancer in a subject, the methodcomprising administering a labeled anti-LgR5 antibody to a subjecthaving or suspected of having an LgR5-positive cancer, and detecting thelabeled anti-LgR5 antibody in the subject, wherein detection of thelabeled anti-LgR5 antibody indicates an LgR5-positive cancer in thesubject. In certain of such embodiments, the labeled anti-LgR5 antibodycomprises an anti-LgR5 antibody conjugated to a positron emitter, suchas ⁶⁸Ga, ¹⁸F, ⁶⁴Cu, ⁸⁶Y, ⁸⁹Zr, and ¹²⁴I. In a particular embodiment, thepositron emitter is ⁸⁹Zr.

In further embodiments, a method of diagnosis or detection comprisescontacting a first anti-LgR5 antibody immobilized to a substrate with abiological sample to be tested for the presence of LgR5, exposing thesubstrate to a second anti-LgR5 antibody, and detecting whether thesecond anti-LgR5 is bound to a complex between the first anti-LgR5antibody and LgR5 in the biological sample. A substrate may be anysupportive medium, e.g., glass, metal, ceramic, polymeric beads, slides,chips, and other substrates. In certain embodiments, a biological samplecomprises a cell or tissue (e.g., biopsy material, including cancerousor potentially cancerous colon, colorectal, small intestine,endometrial, pancreatic or ovarian tissue). In certain embodiments, thefirst or second anti-LgR5 antibody is any of the antibodies describedherein. In some such embodiments, the second anti-LgR5 antibody may be8E11 or antibodies derived from 8E11, e.g., as described herein. In somesuch embodiments, the second anti-LgR5 antibody may be YW353 orantibodies derived from YW353, e.g., as described herein. In someembodiments, the first or second anti-LgR5 antibody is selected from3G12 and 2H6 and antibodies derived from 3G12 and/or 2H6, e.g., asdescribed herein.

Exemplary disorders that may be diagnosed or detected according to anyof the above embodiments include LgR5-positive cancers, such asLgR5-positive colorectal cancer (including adenocarcinoma),LgR5-positive small intestine cancer (including adenocarcinoma, sarcoma(e.g., leiomyosarcoma), carcinoid tumors, gastrointestinal stromaltumor, and lymphoma) LgR5-positive ovarian cancer (including ovarianserous adenocarcinoma), LgR5-positive pancreatic cancer (includingpancreatic ductal adenocarcinoma), and LgR5-positive endometrial cancer.In some embodiments, an LgR5-positive cancer is a cancer that receivesan anti-LgR5 immunohistochemistry (IHC) or in situ hybridization (ISH)score greater than “0,” which corresponds to very weak or no stainingin >90% of tumor cells, under the conditions described herein in ExampleB. In another embodiment, an LgR5-positive cancer expresses LgR5 at a1+, 2+ or 3+ level, as defined under the conditions described herein inExample B. In some embodiments, an LgR5-positive cancer is a cancer thatexpresses LgR5 according to a reverse-transcriptase PCR(RT-PCR) assaythat detects LgR5 mRNA. In some embodiments, the RT-PCR is quantitativeRT-PCR.

In certain embodiments, labeled anti-LgR5 antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like. In anotherembodiment, a label is a positron emitter. Positron emitters include butare not limited to ⁶⁸Ga, ¹⁸F, ⁶⁴Cu, ⁸⁶Y, ⁷⁶Br, ⁸⁹Zr, and ¹²⁴I. In aparticular embodiment, a positron emitter is ⁸⁹Zr.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-LgR5 antibody or immunoconjugateas described herein are prepared by mixing such antibody orimmunoconjugate having the desired degree of purity with one or moreoptional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody or immunoconjugate formulations aredescribed in U.S. Pat. No. 6,267,958. Aqueous antibody orimmunoconjugate formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations including ahistidine-acetate buffer.

The formulation herein may also contain more than one active ingredientas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, in some instances, it may be desirable to furtherprovide Avastin® (bevacizumab), e.g., for the treatment of LgR5-positivecancer such as LgR5-positive colon cancer or LgR5-positive colorectalcancer.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody or immunoconjugate, whichmatrices are in the form of shaped articles, e.g. films, ormicrocapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-LgR5 antibodies or immunoconjugates provided herein maybe used in methods, e.g., therapeutic methods.

In one aspect, an anti-LgR5 antibody or immunoconjugate provided hereinis used in a method of inhibiting proliferation of an LgR5-positivecell, the method comprising exposing the cell to the anti-LgR5 antibodyor immunoconjugate under conditions permissive for binding of theanti-LgR5 antibody or immunoconjugate to LgR5 on the surface of thecell, thereby inhibiting the proliferation of the cell. In certainembodiments, the method is an in vitro or an in vivo method. In furtherembodiments, the cell is a colon, colorectal, small intestine, ovarian,pancreatic, or endometrial cell.

In some embodiments, an anti-LgR5 antibody or immunoconjugate providedherein is used in a method of treating cancer that comprises a mutationin a Kras gene and/or a mutation in an adenomatous polyposis coli (APC)gene in at least a portion of the cells of the cancer. In variousembodiments, the cancer is selected from colon, colorectal, smallintestine, ovarian, pancreatic, and endometrial cancer. In someembodiments, an anti-LgR5 antibody or immunoconjugate provided herein isused in a method of treating a colon or colorectal cancer that comprisesa mutation in a Kras gene and/or a mutation in an APC gene in at least aportion of the cells of the cancer. Nonlimiting exemplary Kras mutationsfound in cancers (including colon and colorectal cancers) includemutations at Kras codon 12 (e.g., G12D, G12V, G12R, G12C, G12S, andG12A), codon 13 (e.g., G13D and G13C), codon 61 (e.g., G61H, G61L, G61E,and G61K), and codon 146. See, e.g., Yokota, Anticancer Agents Med.Chem., 12: 163-171 (2012); Wicki et al., Swiss Med. Wkly, 140: w13112(2010). Nonlimiting exemplary APC mutations found in cancers includemutations in the mutation cluster region (MCR), such as stop codons andframeshift mutations that result in a truncated APC gene product. See,e.g., Chandra et al., PLoS One, 7: e34479 (2012); and Kohler et al.,Hum. Mol. Genet., 17: 1978-1987 (2008).

In some embodiments, a method of treating cancer comprises administeringan anti-LgR5 antibody or immunoconjugate to a subject, wherein thesubject has a cancer comprising a Kras mutation and/or an APC mutationin at least a portion of the cancer cells. In some embodiments, thecancer is selected from colon, colorectal, small intestine, ovarian,pancreatic, and endometrial cancer. In some embodiments, the cancer iscolon and/or colorectal cancer. In some embodiments, the subject haspreviously been determined to have a cancer comprising a Kras mutationand/or an APC mutation in at least a portion of the cancer cells. Insome embodiments, the cancer is LgR5-positive.

Presence of various biomarkers in a sample can be analyzed by a numberof methodologies, many of which are known in the art and understood bythe skilled artisan, including, but not limited to, immunohistochemistry(“IHC”), Western blot analysis, immunoprecipitation, molecular bindingassays, ELISA, ELIFA, fluorescence activated cell sorting (“FACS”),MassARRAY, proteomics, quantitative blood based assays (as for exampleSerum ELISA), biochemical enzymatic activity assays, in situhybridization, Southern analysis, Northern analysis, whole genomesequencing, polymerase chain reaction (“PCR”) including quantitativereal time PCR (“qRT-PCR”) and other amplification type detectionmethods, such as, for example, branched DNA, SISBA, TMA and the like,RNA-Seq, FISH, microarray analysis, gene expression profiling, and/orserial analysis of gene expression (“SAGE”), as well as any one of thewide variety of assays that can be performed by protein, gene, and/ortissue array analysis. Typical protocols for evaluating the status ofgenes and gene products are found, for example in Ausubel et al., eds.,1995, Current Protocols In Molecular Biology, Units 2 (NorthernBlotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCRAnalysis). Multiplexed immunoassays such as those available from RulesBased Medicine or Meso Scale Discovery (“MSD”) may also be used.

Inhibition of cell proliferation in vitro may be assayed using theCellTiter-Glo™ Luminescent Cell Viability Assay, which is commerciallyavailable from Promega (Madison, Wis.). That assay determines the numberof viable cells in culture based on quantitation of ATP present, whichis an indication of metabolically active cells. See Crouch et al. (1993)J. Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677. The assay may beconducted in 96- or 384-well format, making it amenable to automatedhigh-throughput screening (HTS). See Cree et al. (1995) AntiCancer Drugs6:398-404. The assay procedure involves adding a single reagent(CellTiter-Glo® Reagent) directly to cultured cells. This results incell lysis and generation of a luminescent signal produced by aluciferase reaction. The luminescent signal is proportional to theamount of ATP present, which is directly proportional to the number ofviable cells present in culture. Data can be recorded by luminometer orCCD camera imaging device. The luminescence output is expressed asrelative light units (RLU).

In another aspect, an anti-LgR5 antibody or immunoconjugate for use as amedicament is provided. In further aspects, an anti-LgR5 antibody orimmunoconjugate for use in a method of treatment is provided. In certainembodiments, an anti-LgR5 antibody or immunoconjugate for use intreating LgR5-positive cancer is provided. In certain embodiments, theinvention provides an anti-LgR5 antibody or immunoconjugate for use in amethod of treating an individual having an LgR5-positive cancer, themethod comprising administering to the individual an effective amount ofthe anti-LgR5 antibody or immunoconjugate. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, e.g., as describedbelow.

In a further aspect, the invention provides for the use of an anti-LgR5antibody or immunoconjugate in the manufacture or preparation of amedicament. In one embodiment, the medicament is for treatment ofLgR5-positive cancer. In a further embodiment, the medicament is for usein a method of treating LgR5-positive cancer, the method comprisingadministering to an individual having LgR5-positive cancer an effectiveamount of the medicament. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, e.g., as described below.

In a further aspect, the invention provides a method for treatingLgR5-positive cancer. In one embodiment, the method comprisesadministering to an individual having such LgR5-positive cancer aneffective amount of an anti-LgR5 antibody or immunoconjugate. In onesuch embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, as described below.

An LgR5-positive cancer according to any of the above embodiments maybe, e.g., LgR5-positive colon or colorectal cancer (includingadenocarcinoma), LgR5-positive small intestine cancer (includingadenocarcinoma, sarcoma (e.g., leiomyosarcoma), carcinoid tumors,gastrointestinal stromal tumor, and lymphoma)., LgR5-positive ovariancancer (including ovarian serous adenocarcinoma), LgR5-positivepancreatic cancer (including pancreatic ductal adenocarcinoma), andLgR5-positive endometrial cancer. In some embodiments, an LgR5-positivecancer is a cancer that receives an anti-LgR5 immunohistochemistry (IHC)or in situ hybridization (ISH) score greater than “0,” which correspondsto very weak or no staining in >90% of tumor cells, under the conditionsdescribed herein in Example B. In another embodiment, an LgR5-positivecancer expresses LgR5 at a 1+, 2+ or 3+ level, as defined under theconditions described herein in Example B. In some embodiments, anLgR5-positive cancer is a cancer that expresses LgR5 according to areverse-transcriptase PCR(RT-PCR) assay that detects LgR5 mRNA. In someembodiments, the RT-PCR is quantitative RT-PCR.

An “individual” according to any of the above embodiments may be ahuman.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-LgR5 antibodies or immunoconjugate providedherein, e.g., for use in any of the above therapeutic methods. In oneembodiment, a pharmaceutical formulation comprises any of the anti-LgR5antibodies or immunoconjugates provided herein and a pharmaceuticallyacceptable carrier. In another embodiment, a pharmaceutical formulationcomprises any of the anti-LgR5 antibodies or immunoconjugates providedherein and at least one additional therapeutic agent, e.g., as describedbelow.

Antibodies or immunoconjugates of the invention can be used either aloneor in combination with other agents in a therapy. For instance, anantibody or immunoconjugate of the invention may be co-administered withat least one additional therapeutic agent. In certain embodiments, anadditional therapeutic agent is Avastin® (bevacizumab), e.g., for thetreatment of LgR5-positive cancer such as LgR5-positive colon cancer orLgR5-positive colorectal cancer.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody or immunoconjugate of the invention canoccur prior to, simultaneously, and/or following, administration of theadditional therapeutic agent and/or adjuvant. Antibodies orimmunoconjugates of the invention can also be used in combination withradiation therapy.

An antibody or immunoconjugate of the invention (and any additionaltherapeutic agent) can be administered by any suitable means, includingparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies or immunoconjugates of the invention would be formulated,dosed, and administered in a fashion consistent with good medicalpractice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The antibody or immunoconjugate need notbe, but is optionally formulated with one or more agents currently usedto prevent or treat the disorder in question. The effective amount ofsuch other agents depends on the amount of antibody or immunoconjugatepresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody or immunoconjugate of the invention (when used alone or incombination with one or more other additional therapeutic agents) willdepend on the type of disease to be treated, the type of antibody orimmunoconjugate, the severity and course of the disease, whether theantibody or immunoconjugate is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody or immunoconjugate, and the discretion ofthe attending physician. The antibody or immunoconjugate is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody or immunoconjugate can be aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. One typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs. One exemplary dosage of the antibody orimmunoconjugate would be in the range from about 0.05 mg/kg to about 10mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kgor 10 mg/kg (or any combination thereof) may be administered to thepatient. Such doses may be administered intermittently, e.g. every weekor every three weeks (e.g. such that the patient receives from about twoto about twenty, or e.g. about six doses of the antibody). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using both an immunoconjugate of theinvention and an anti-LgR5 antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thedisorder and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody or immunoconjugate of the invention. Thelabel or package insert indicates that the composition is used fortreating the condition of choice. Moreover, the article of manufacturemay comprise (a) a first container with a composition contained therein,wherein the composition comprises an antibody or immunoconjugate of theinvention; and (b) a second container with a composition containedtherein, wherein the composition comprises a further cytotoxic orotherwise therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition. Alternatively, or additionally, the article of manufacturemay further comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution ordextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

A. Human LgR5 Gene Expression

Human LgR5 gene expression was analyzed using a proprietary databasecontaining gene expression information (GeneExpress®, Gene Logic Inc.,Gaithersburg, Md.). Graphical analysis of the GeneExpress® database wasconducted using a microarray profile viewer. FIG. 1 is a graphicrepresentation of human LgR5 gene expression in various tissues. Thescale on the y-axis indicates gene expression levels based onhybridization signal intensity. Dots appear both to the left and to theright of the line extending from the name of each listed tissue. Thedots appearing to the left of the line represent gene expression innormal tissue, and the dots appearing to the right of the line representgene expression in tumor and diseased tissue. FIG. 1 shows increasedLgR5 gene expression in certain tumor or diseased tissues relative totheir normal counterparts. In particular, LgR5 is substantiallyoverexpressed in colorectal, endometrial, and ovarian tumors. FIG. 1,inset, shows that LgR5 is overexpressed in at least the following colontumors: adenocarcinoma, benign tumors, and metastatic colon tumors, andalso in tissue with a colon tumor content of less than 50% (“low tumor”in FIG. 1 inset); but is not overexpressed in normal colon, Crohn'sdisease, or ulcerative colitis. Human LgR5 expression is much lower innormal tissues, with low levels of expression in normal brain, muscle,ovarian, and placental tissues.

B. Prevalence of Human LgR5 in Colon Tumors

To evaluate the expression of LgR5 in colorectal cancer, 57 primarycolorectal adenocarcinomas were acquired from multiple sources(Asterand, Detroit, Mich.; Bio-Options, Fullerton, Calif.; University ofMichigan, Ann Arbor, Mich.; Cytomyx, Rockville, Md.; Cooperative HumanTissue Network, Nashville, Tenn.; Indivumed, Hamburg, Germany;ProteoGenex, Culver City, Calif.). Forty-four percent of samples werefrom men, and the average age of the patients was 66 years (range 31 to93 years). Tissue microarrays (TMAs) were assembled using duplicatecores as described in Bubendorf L, et al., J Pathol. 2001 September;195(1):72-9, and included five normal colorectal mucosa samples frommatched cases.

LgR5 expression was determined by in situ hybridization using theoligonucleotide probes shown in Table 2. See, e.g., Jubb A M, et al.,Methods Mol Biol 2006; 326:255-64. ISH for β-actin was used to confirmmRNA integrity in colorectal cancer tissues prior to analysis.

TABLE 2 Primer sequences for isotopic in situ hybridization probes.Anti- Nucleotides sense Comple- (AS) or Genbank mentary to SenseForward Primer (5′ to Gene Accession Probe (S) 3′) Reverse Primer (5′to 3′) Lgr5 NM_003667 508 AS ACCAACTGCATCCT ACCGAGTTTCACCTCAAACTG (SEQ ID NO: AGCTC (SEQ ID NO: 83) 84) Lgr5 NM_003667 496 SACATTGCCCTGTTGC ACTGCTCTGATATAC TCTTC (SEQ ID NO: TCAATC (SEQ ID NO: 85)86)

LgR5 hybridization intensity was scored by a trained pathologistaccording to the scheme below, taking into account the intensity (silvergrains) as well as breadth of staining.

-   -   0 (negative): very weak or no hybridization in >90% of tumor        cells    -   1+ (mild): predominant hybridization pattern is weak    -   2+ (moderate): predominant hybridization pattern is moderately        strong in the majority (>50%) of neoplastic cells    -   3+ (strong): predominant hybridization pattern is strong in the        majority (>50%) of neoplastic cells        Sense probes were used to control for the specificity of        hybridization.

FIG. 2 shows exemplary colon tumor sections with 1+, 2+, and 3+ levelsof staining. The top panels show dark field images and the bottom panelsshow bright field images. The deposition of silver grains in the darkfield images indicates hybridization of the probe and expression of LgR5mRNA. ˜77% (41/53) of colon tumor sections analyzed were LgR5 positive,showing staining at the 1+, 2+, or 3+ levels, with 34% (18/53) showing2+ or 3+ staining. Four of the 57 samples analyzed were noninformativefor LgR5 expression.

To evaluate the significance of Lgr5 expression in colon tumors, apopulation-based series of patients who had undergone surgicalresections for colorectal adenocarcinoma was compiled retrospectivelyfrom the pathology archives at St James' University Hospital (Leeds, UK)from 1988 to 2003. Tissue microarrays (TMAs) were constructed with onecore of normal mucosa and three cores of adenocarcinoma per patient asdescribed in Bubendorf L, et al., J Pathol. 2001 September; 195(1):72-9.ISH was performed and scored as described above. The heterogeneity ofexpression across three cores from the same tumor was also determined,and is expressed as the proportion of tumors that showed a particularlevel of discordance in one of the three cores. For example, if threecores had scores of +1, +3, and +3, one of the three cores from thattumor is discordant by 2.

FIG. 3A shows the prevalence of 0, 1+, 2+, and 3+ levels of LgR5staining in the colon tumor tissue microarray, measured by in situhybridization. 75% of the colon tumor tissues showed staining at the 1+,2+, or 3+ levels, with 37% showing 2+ or 3+ staining. FIG. 3B shows theheterogeneity of LgR5 expression. 67% of tumors showed no heterogeneityacross the three cores. 32% shows a discordance of 1 in one of the threecores, and only 1% showed a discordance greater than 1.

C. Mouse Monoclonal Antibody Generation

Monoclonal antibodies against human LgR5 were generated using thefollowing procedures. Human LgR5 extracellular domain (ECD; amino acids22-557) with a C-terminal His-tagged Fc was expressed from a baculovirusexpression system, and purified on a Ni-NTA column (Qiagen), followed bygel filtration on a Superdex 200 column in 20 mM MES pH 6.0, 6Mguanidine HCl as previously described (Kirchhofer et al., 2003) anddialysis into PBS for storage at −80° C.

Fifteen Balb/c mice (Charles River Laboratories International, Inc.,Hollister, Calif., USA) were injected with either huLgR5 plasmid DNA inlactated Ringer's solution (via tail vein) or with recombinant humanLgR5 ECD as described above (via rear footpads) in adjuvant containingmetabolizable squalene (4% v/v), Tween 80 (0.2% v/v), trehalose6,6-dimycolate (0.05% w/v) and monophosphoryl lipid A (0.05% w/v; SigmaAldrich, USA). Serum titers were evaluated by standard enzyme linkedimmunosorbant assay (ELISA) and FACS following 6-9 injections. Splenic Bcells harvested from a total of 5 mice were fused with mouse myelomacells (X63.Ag8.653; American Type Culture Collection, Manassas, Va.,USA) by electrofusion (Hybrimune; Harvard Apparatus, Inc., Holliston,Mass., USA). After 10-14 days, hybridoma supernatants were screened forantibody secretion by ELISA. All positive clones were then expanded andre-screened for binding to huLgR5 and muLgR5 by ELISA and FACS (i.e.,for binding to 293-huLGR5 and 293-muLGR5 cells). Hybridoma clones8E11.1.1 (identified from the DNA immunized mice), and 2H6.3.5 and3G12.2.1 (both from the protein immunized mice) showed highimmunobinding after two rounds of subcloning (by limiting dilution) andwere scaled up for purification in INTEGRA CELLine 1000 bioreactors(INTEGRA Biosciences AG, Zizers, Switzerland). Supernatants were thenpurified by affinity chromatography, sterile-filtered, and stored at 4°C. in PBS. The isotypes of the mAbs were determined to be IgG1 (kappalight chain) using the Isostrip Mouse mAb Isotyping Kit (Roche AppliedBiosciences, Indianapolis, Ind., USA).

FIG. 4 shows certain monoclonal antibodies generated, along with certainproperties, some of which will be described in further detail below.

D. Cloning and Chimerization of Mouse Monoclonal Antibodies

Monoclonal antibodies 8E11, 3G12, and 2H6 were cloned and chimerized asfollows.

Total RNA was extracted from hybridoma cells producing murine 8E11,murine 3G12, or murine 2H6 using standard methods. The variable light(VL) and variable heavy (VH) domains were amplified using RT-PCR withdegenerate primers to the heavy and light chains. The forward primerswere specific for the N-terminal amino acid sequence of the VL and VHregions. Respectively, the LC and HC reverse primers were designed toanneal to a region in the constant light (CL) and constant heavy domain1 (CH1), which are highly conserved across species. The polynucleotidesequence of the inserts was determined using routine sequencing methods.The 8E11 VL and VH amino acid sequences are shown in FIGS. 5 and 6,respectively (SEQ ID NOs: 3 and 4, respectively). The 3G12 and 2H6 VLand VH amino acid sequences are shown in FIGS. 7 and 8, respectively.The VL and VH sequences of antibody 3G12 are shown in SEQ ID NOs: 21 and22, respectively, and the VL and VH sequences of antibody 2H6 are shownin SEQ ID NOs: 23 and 24, respectively.

Each antibody was chimerized by cloning the mouse heavy chain variableregion onto a human IgG1 heavy chain constant region and cloning thelight chain variable region onto a human kappa light chain constantregion.

E. Humanization of 8E11

Monoclonal antibody 8E11 was humanized as described below. Residuenumbers are according to Kabat et al., Sequences of proteins ofimmunological interest, 5th Ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).

Direct Hypervariable Region Grafts onto the Acceptor Human ConsensusFramework

Variants constructed during the humanization of 8E11 were assessed inthe form of an IgG. The VL and VH domains from murine 8E11 were alignedwith the human VL kappa IV (VL_(KIV)) and human VH subgroup I (VH_(I))consensus sequences. Hypervariable regions from the murine 8E11 (mu8E11)antibody were engineered into VL_(KIV) and VH_(I) acceptor frameworks togenerate 8E11.v1. Specifically, from the mu8E11 VL domain, positions24-34 (L1), 50-56 (L2) and 89-97 (L3) were grafted into VL_(KIV). Fromthe mu8E11 VH domain, positions 26-35 (H1), 49-65 (H2) and 95-102 (H3)were grafted into VH_(I). In addition, positions 71 and 78 in frameworkIII of VH were retained from the mouse sequence in 8E11.v1. Thoseresidues were found to be part of the framework residues acting as“Vernier” zone, which may adjust CDR structure and fine-tune the antigenfit. See, e.g., Foote and Winter, J. Mol. Biol. 224: 487-499 (1992)(FIGS. 5 and 6). These CDR definitions include positions defined bytheir sequence hypervariability (Wu, T. T. & Kabat, E. A. (1970)), theirstructural location (Chothia, C. & Lesk, A. M. (1987)) and theirinvolvement in antigen-antibody contacts (MacCallum et al. J. Mol. Biol.262: 732-745 (1996)).

Additional 8E11 variants were generated to evaluate the contributions ofother Vernier positions, such as position 68 in the light chain, andpositions 67 and 69 in the heavy chain. Humanized 8E11.v2 was generatedby retaining two addition mouse residues, at positions 67 and 69 of theheavy chain variable region. The light chain variable region sequenceand heavy chain variable region sequence for 8E11.v2, and othervariants, are shown in FIGS. 5 and 6, respectively.

The humanized variants of 8E11 were generated by Kunkel mutagenesisusing a separate oligonucleotide for each hypervariable region. Correctclones were identified by DNA sequencing.

Assessment of Variants

For screening purposes, IgG variants were initially produced in 293cells. Vectors coding for VL and VH were transfected into 293 cells. IgGwas purified from cell culture media by protein A affinitychromatography.

The affinity of each 8E11 IgG variant for human LgR5 was determined bysurface plasmon resonance using a BIAcore™-3000. BIAcore™ research gradeCM5 chips were activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) reagents according tothe supplier's instructions. Goat anti-human Fc IgGs were coupled to thechips to achieve approximately 10,000 response units (RU) in each flowcell. Unreacted coupling groups were blocked with 1M ethanolamine. Forkinetics measurements, anti-LGR5 antibodies were captured to achieveapproximately 300 RU. Two-fold serial dilutions of human LgR5 ECD (aminoacids 22-557 fused to His-Fc expressed in a baculovirus system, or aminoacids 22-558 fused to Fc expressed from CHO cells; 125 nM to 0.49 nM)were injected in HBS-P buffer (0.01M HEPES pH7.4, 0.15M NaCl, 0.005%surfactant P20) at 25° C. with a flow rate of 30 μl/min. Associationrates (k_(on)) and dissociation rates (k_(off)) were calculated using a1:1 Langmuir binding model (BIAcore™ Evaluation Software version 3.2).The equilibrium dissociation constant (Kd) was calculated as the ratiok_(off)/k_(on).

Results

The human acceptor framework used for humanization of 8E11 is based onthe human VL kappa IV consensus (VL_(KIV)) and the acceptor VH frameworkVH_(I). Eight humanized variants of mu8E11 were produced and tested forLgR5 affinity by BIAcore™. The light chain variable regions and heavychain variable regions of each of the variants is shown in FIGS. 5 and6, respectively. The results of the affinity measurements are shown inFIG. 9.

To improve the binding affinity of 8E11.v1, position 68 in the lightchain and positions of 67 and 69 in the heavy chain were changed toresidues found at these positions in mu8E11. Positions 71 and 78 in theheavy chain were changed to residues found at these positions in thehuman framework VH_(I). Combinations of these altered light and heavychains were expressed as IgG and purified as described above, andassessed for binding to human LgR5 by Biacore (FIG. 9).

Variant hu8E11.v2 was generated by changing positions 67 and 69 of thehu8E11.v1 heavy chain to the residues found at those positions inmu8E11. The affinity (K_(D)) of hu8E11.v2 was found to be about the sameas the parental ch8E11 antibody.

Summary of Changes for Humanized 8E11.v2

The 6 murine 8E11 CDRs (defined as positions 24-34 (L1), 50-56 (L2) and89-97 (L3), 26-35 (H1), 49-65 (H2) and 93-102 (H3)) were grafted intothe human consensus VL_(KIV), and VH_(I) acceptor domains. Positions 67,69, 71, and 78 were changed back to murine residues from mu8E11.Humanized 8E11.v2 has comparable affinity for LgR5 to chimeric 8E11.

Throughout this application, mouse monoclonal antibodies 8E11, 2H6, and3G12 are referred to in the alternative as 8E11, m8E11, or mu8E11; and2H6, m2H6 or mu2H6; and 3G12, m3G12, or mu3G12; respectively. Chimericmonoclonal antibodies 8E11, 2H6, and 3G12 are referred to as chimeric8E11 or ch8E11; chimeric 2H6 or ch2H6; and chimeric 3G12 or ch3G12;respectively. Humanized monoclonal antibody 8E11.v2 may also be referredto as 8E11v2, h8E11v.2, or hu8E11v.2.

F. Generation of a Human Monoclonal Antibody by Phage Display

Human LgR5 ECD (amino acids 22-555) with an N-terminal FLAG wasexpressed in CHO cells and purified on an anti-FLAG resin overnight, andthen eluted with 0.1M acetic acid, pH 2.7. The protein was then purifiedby gel filtration on a Superdex 200 column in PBS and then dialyzed intoPBS for storage at −80° C.

Human phage antibody libraries with synthetic diversities in theselected complementary determining regions (H1, H2, H3), mimicking thenatural diversity of human IgG repertoire were used for panning. The Fabfragments were displayed bivalently on the surface of M13 bacteriophageparticals (Lee et al. (2004) J Mol Biol 340, 1073-93). Human LgR5 ECD(amino acids 22-555) produced as described above was used as an antigen.Nunc 96-well MaxiSorp immunoplates (Nunc) were coated overnight at 4° C.with LgR5 ECD protein (10 μg/ml) and blocked for 1 hour with PBST buffer(PBS, 0.05% Tween 20) supplemented with 1% BSA. The antibody phagelibraries were added and incubated overnight at room temperature. Theplates were washed with PBST buffer and bound phage were eluted with 50mM HCL/500 mM NaCl for 30 minutes and neutralized with an equal volumeof 1M Tris base. Recovered phages were amplified in E. coli XL-1 bluecells. During subsequent selection rounds, the incubation time of thephage antibodies was decreased to 2 hours and the stringency of platewashing was gradually increased (Liang et al. (2007) J Mol Biol 366,815-829). Unique and specific phage antibodies that bind to human LgR5ECD were identified by phage ELISA and DNA sequencing. Certain clones,including YW353, were reformatted to full length IgGs by cloning the VLand VH regions into LPG3 and LPG4 vectors, respectively. Antibodies weretransiently expressed in mammalian cells and purified on protein Acolumns (Carter et al. (1992) Proc Natl Acad Sci USA 89, 4285-9).

The light chain and heavy chain variable regions sequence for humanantibody YW353 are shown in FIGS. 10 and 11, respectively (SEQ ID NOs:26 and 25). IgG1 heavy chain and kappa light chain sequences for humanantibody YW353 are shown in SEQ ID NOs: 66 and 65, respectively. SinceYW353 was generated from a human antibody phage library, the terms“YW353” and “huYW353” are used interchangeably herein.

G. Species Cross-Reactivity

Monoclonal antibodies were tested to determine if they cross-react withLgR5 from species other than human. FIGS. 12A to C shows an alignmentbetween human (SEQ ID NO: 67), cynomolgus monkey (SEQ ID NO: 69), rat(SEQ ID NO: 70) and mouse (SEQ ID NO: 72) LgR5. Residues that areidentical among all four species are indicated by asterisks (*). FIG. 4shows the results of FACS analysis of 293 cells stably transfected withgD epitope-tagged LgR5 (human, cynomolgus monkey, rat, or mouse LgR5);stained with 10 μg/ml YW353, ch8E11, hu8E11.v2, 2H6, or 3G12 antibody;and detected with R-Phycoerythrin conjugated goat anti-human antibody.Untransfected 293 cells do not normally express LgR5. YW353 antibodybinds human and cynomolgus monkey LgR5, but not rat or mouse LgR5.Ch8E11 and hu8E11.v2 antibodies bind all four species of LgR5, althoughbinding to rat LgR5 is not as strong as binding to human, cynomolgusmonkey, or mouse LgR5. 2H6 antibody binds to human and mouse LgR5, andwas not tested for binding to cynomolgus monkey or rat LgR5. 3G12antibody shows strong binding to human LgR5, less strong binding tomouse LgR5, and was not tested for binding to cynomolgus monkey or ratLgR5.

H. Antibody Affinities

The affinity of each antibody for human LgR5 was determined by surfaceplasmon resonance using a BIAcore™-3000, substantially as describedabove in Example E.

As shown in FIG. 4, YW353 antibody bound to human LgR5 with an affinityof 1.6 nM. Ch8E11 and hu8E11.v2 antibodies bound to human LgR5 withaffinities of 2.4 nm and 3.1 nm, respectively. 2H6 and 3G12 antibodiesbound to human LgR5 with affinities of 208 nM and 72 nM, respectively.

Scatchard analysis was performed following standard procedures (Holmeset al., Science 256:1205-1210 (1992)) to determine the relative bindingaffinities of YW353, ch8E11 and hu8E11v2 antibodies.

Anti-Lgr5 antibodies were [I¹²⁵] labeled using the indirect Iodogenmethod. The [I¹²⁵] labeled anti-Lgr5 antibodies were purified from free¹²⁵I-Na by gel filtration using a NAP-5 column (GE Healthcare); thepurified iodinated anti-Lgr5 antibodies had a range of specificactivities of 13.92 to 19.01 μCi/μg. Competition assay mixtures of 50 μLvolume containing a fixed concentration of [I¹²⁵] labeled antibody anddecreasing concentrations of serially diluted, unlabeled antibody wereplaced into 96-well plates. 293 cells stably expression human, rat, ormouse Lgr5 were cultured in growth media at 37° C. in 5% CO₂. Cells weredetached from the flask using Sigma Cell Dissociation Solution and werewashed with binding buffer, which consisted of Dulbecco's Modified EagleMedium (DMEM) with 2% fetal bovine serum (FBS), 50 mM HEPES (pH 7.2) and0.1% sodium azide. The washed cells were added to the 96 well plates ata density of 250,000 cells in 0.2 mL of binding buffer. The finalconcentration of the labeled antibody in each well was 200 μM. The finalconcentration of the unlabeled antibody in the competition assay rangedfrom 500 nM through ten 2-fold dilution steps to a 0 nM buffer-onlyassay. Competition assays were carried out in triplicate. Competitionassays were incubated for 2 hours at room temperature. After the 2-hourincubation, the competition assays were transferred to a MilliporeMultiscreen filter plate (Billerica, Mass.) and washed 4 times withbinding buffer to separate the free from bound [I¹²⁵] labeled antibody.The filters were counted on a Wallac Wizard 1470 gamma counter(PerkinElmer Life and Analytical Sciences Inc.; Wellesley, Mass.). Thebinding data was evaluated using NewLigand software (Genentech), whichuses the fitting algorithm of Munson and Robard to determine the bindingaffinity of the antibody (Munson and Robard 1980)

As shown in FIG. 4, YW353 bound to gD-tagged human LgR5 expressed onstably transfected 293 cells with an affinity of 0.2 nM. Ch8E11 bound togD-tagged human LgR5 and gD-tagged mouse LgR5 expressed on stablytransfected 293 cells with affinities of 0.4 nM and 0.2 nM,respectively. Hu8E11v2 bound to gD-tagged human LgR5, gD-tagged mouseLgR5, and gD-tagged rat LgR5 expressed on stably transfected 293 cellswith affinities 0.3-0.7 nM, 0.5-0.6 nM, and 2.4-2.8 nM, respectively.These Kd values were generally lower than those determined by BIAcore®.

I. Epitope Mapping

To determine the region of LgR5 bound by each antibody, 293 cellstransiently transfected with gD epitope-tagged LgR5 with various N-and/or C-terminal deletions were stained with 10 μg/ml YW353, ch8E11,hu8E11v2, 2H6, or 3G12 antibody; and binding was detected withR-Phycoerythrin conjugated goat anti-human antibody. Antibodies YW353,8E11, 2H6, and 3G12 all bound to gD epitope-tagged full-length LgR5.Antibodies 2H6 and 3G12 bound to gD epitope-tagged LgR5₃₂₄₋₉₀₇ (aminoacids 324-907 of SEQ ID NO: 67). Antibodies YW353 and 8E11 did not bindto gD epitope-tagged LgR5₃₂₄₋₉₀₇. Only antibody YW353 bound to gDepitope-tagged LgR5₂₂₋₁₂₃ (amino acids 22-123 of SEQ ID NO: 67) with aC-terminal GPI anchor. Antibodies YW353 and 8E11 both bound to gDepitope-tagged LgR5₂₂₋₃₂₃ (amino acids 22-323 of SEQ ID NO: 67) with aC-terminal GPI anchor, but antibodies 2H6 and 3G12 did not. Finally,none of the antibodies bound to gD epitope-tagged LgR5₄₂₄₋₉₀₇ (aminoacids 424-907 of SEQ ID NO: 67).

FIG. 4 summarizes those results in the column titled “epitope region.”As shown in that figure, antibody YW353 binds to an epitope in theregion of amino acids 22 to 123 of SEQ ID NO: 67; antibody 8E11 and itshumanized variants bind to an epitope in the region of amino acids 22 to323 of SEQ ID NO: 67; and antibodies 2H6 and 3G12 bind to an epitope inthe region of amino acids 324 to 423 of SEQ ID NO: 67.

J. Production of Anti-LgR5 Antibody Drug Conjugates

For larger scale antibody production, antibodies were produced in CHOcells. Vectors coding for VL and VH were transfected into CHO cells andIgG was purified from cell culture media by protein A affinitychromatography.

Anti-LgR5 Antibody MMAE Conjugates

Anti-LgR5 antibody-drug conjugates (ADCs) were produced by conjugatingYW353 (IgG1 heavy chain and kappa light chain sequences shown in SEQ IDNOs: 66 and 65, respectively), hu8E11v2 (IgG1 heavy chain and kappalight chain sequences shown in SEQ ID NOs: 64 and 63, respectively),mu8E11, ch8E11, 2H6, ch2H6, 3G12, and ch3G12 to the drug-linker moietyMC-vc-PAB-MMAE, which is depicted herein. For convenience, thedrug-linker moiety MC-vc-PAB-MMAE is sometimes referred to in theseExamples and in the Figures as “vcMMAE” or “VCE.” Prior to conjugation,the antibodies were partially reduced with TCEP using standard methodsin accordance with the methodology described in WO 2004/010957 A2. Thepartially reduced antibodies were conjugated to the drug-linker moietyusing standard methods in accordance with the methodology described,e.g., in Doronina et al. (2003) Nat. Biotechnol. 21:778-784 and US2005/0238649 A1. Briefly, the partially reduced antibodies were combinedwith the drug-linker moiety to allow conjugation of the drug-linkermoiety to reduced cysteine residues of the antibody. The conjugationreactions were quenched, and the ADCs were purified. The drug load(average number of drug moieties per antibody) for each ADC wasdetermined and was between 3.3 and 4.0 for the anti-LgR5 antibodies. Thestructure of an antibody-vcMMAE immunoconjugate is shown in FIG. 35A(p=drug load).

Anti-LgR5 Antibody PNU Conjugates

Anti-LgR5 antibody-PNU drug conjugates (ADCs) were produced byconjugating YW353 A118C thioMab (IgG1 A118C heavy chain and kappa lightchain sequences shown in SEQ ID NOs: 78 and 65, respectively) orhu8E11v2 thioMab (IgG1 A118C heavy chain and kappa light chain sequencesshown in SEQ ID NOs: 75 and 63, respectively) to PNU drug-linkermoieties. Prior to conjugation, the antibody was reduced withdithiothreitol (DTT) to remove blocking groups (e.g. cysteine) from theengineered cysteines of the thio-antibody. This process also reduces theinterchain disulfide bonds of the antibody. The reduced antibody waspurified to remove the released blocking groups and the interchaindisulfides were reoxidized using dehydro-ascorbic acid (dhAA).

For antibody-drug conjugates comprising a val-cit linker and PNU, theintact antibody was combined with the drug-linker moietyMC-val-cit-PAB-spacer-PNU-159682 (“val-cit” may also be referred toherein as “vc”) to allow conjugation of the drug-linker moiety to theengineered cysteine residues of the antibody. The conjugation reactionwas quenched by adding excess N-acetyl-cysteine to react with any freelinker-drug moiety, and the ADC was purified. The drug load (averagenumber of drug moieties per antibody) for the ADC was in the range ofabout 1.8 to 2. The structure of an antibody-vcPNU immunoconjugate isshown in FIG. 35B (p=drug load).

For antibody drug conjugates comprising an acetal linker and PNU, theintact antibody was combined with the drug-linker moietyMC-acetal-PNU-159682 to allow conjugation of the drug-linker moiety tothe engineered cysteine residues of the antibody. The conjugationreaction was quenched by adding excess N-acetyl-cysteine to react withany free linker-drug moiety, and the ADC was purified. The drug load(average number of drug moieties per antibody) for the ADC was about 1.8to 2. The structure of an antibody-acetal-PNU immunoconjugate is shownin FIG. 35C (p=drug load).

For antibody drug conjugates comprising a noncleavable linker and PNU,the intact antibody was combined with the drug-linker moietyMC-PNU-159682 to allow conjugation of the drug-linker moiety to theengineered cysteine residues of the antibody. The conjugation reactionwas quenched by adding excess N-acetyl-cysteine to react with any freelinker-drug moiety, and the ADC was purified. The drug load (averagenumber of drug moieties per antibody) for the ADC was about 1.8 to 2.The structure of an antibody-PNU immunoconjugate is shown in FIG. 35D(p=drug load).

Anti-LgR5 Antibody PBD Conjugate

Anti-LgR5 antibody-PBD drug conjugates (ADCs) were produced byconjugating YW353 A118C thioMab (IgG1 A118C heavy chain and kappa lightchain sequences shown in SEQ ID NOs: 78 and 65, respectively) orhu8E11v2 thioMab (IgG1 A118C heavy chain and kappa light chain sequencesshown in SEQ ID NOs: 75 and 63, respectively) to PBD drug-linkermoieties. Prior to conjugation, the antibody was reduced withdithiothreitol (DTT) to remove blocking groups (e.g. cysteine) from theengineered cysteines of the thio-antibody. This process also reduces theinterchain disulfide bonds of the antibody. The reduced antibody waspurified to remove the released blocking groups and the interchaindisulfides were reoxidized using dehydro-ascorbic acid (dhAA).

For antibody-drug conjugates comprising a val-cit linker and PBD, theintact antibody was combined with the drug-linker moietyMC-val-cit-PAB-PBD (“val-cit” may also be referred to herein as “vc”) toallow conjugation of the drug-linker moiety to the engineered cysteineresidues of the antibody. The conjugation reaction was quenched byadding excess N-acetyl-cysteine to react with any free linker-drugmoiety, and the ADC was purified. The drug load (average number of drugmoieties per antibody) for the ADC was in the range of about 1.8 to 2.The structure of an antibody-vcPBD is shown in FIG. 35E (p=drug load).

K. Toxicity of Anti-LgR5 Antibody Drug Conjugate in Rats

In order to evaluate potential toxicity of anti-LgR5 antibody8E11.v2-vcMMAE, six male Sprague-Dawley rats were administered 12 mg/kg8E11.v2-vcMMAE once per week for four weeks, and six male Sprague-Dawleyrats were administered 20 mg/kg 8E11.v2-vcMMAE once per week for twoweeks. Four male Sprague-Dawley control rats were administered vehiclealone once per week for two weeks, and four male Sprague-Dawley controlrats were administered vehicle alone once per week for four weeks. Therats in the two-week groups were necropsied on day 12 and the rats inthe four-week groups were necropsied on day 26.

Briefly, all rats administered 8E11.v2-vcMMAE showed reduced red cellmass (red blood cells, hematocrit, hemoglobin, and reticulocytes),neutrophils, and platelets compared to control rats. Rats administered20 mg/kg 8E11.v2-vcMMAE also showed reduced white blood cell count andlymphocytes compared to control rats. In addition, all rats administered8E11.v2-vcMMAE showed increases in liver enzymes ALT, AST, ALP, and GGT,and increased total billirubin compared to control rats.

Histopathologic analysis of tissues collected from the study showedcellular depletion of lymphoid and hemopoietic tissues in ratsadministered 8E11.v2-vcMMAE, as well as increased mitotic figures inrapidly dividing tissues. Rats administered 20 mg/kg 8E11.v2-vcMMAE alsoshowed minimal liver necrosis, minimal increased mitotic figures andsingle cell cryptal necrosis/apoptosis, and minimal mild alveolarhistiocytosis and type II cell hyperplasia.

The pathology changes observed were similar to pathology observed inrats administered other vcMMAE antibody-drug conjugates. There did notappear to be any evidence of LgR5 antigen-dependent toxicity in the GItract.

L. Efficacy of Anti-LgR5 Antibody Drug Conjugates in LoVo Colon CancerCell Line Xenograft

The efficacy of the anti-LgR5 ADCs was investigated using a LoVo coloncancer xenograft model. LoVo cells are a colorectal adenocarcinoma cellline with an APC mutation (ATCC # CCL 229). LgR5 is highly expressed inLoVo cells, and was confirmed by microarray, TaqMan® quantitativeRT-PCR, in situ hybridization, FACS, and Western blot. Five million LoVocells (LgR5-positive by FACS using YW353) in HBSS-matrigel were injectedsubcutaneously into the dorsal flank of NCR nude mice and six dayspost-inoculation mice were given a single intravenous injection of 5mg/kg murine anti-gp120-vcMMAE control antibody-drug conjugate, humananti-gD 5B6-vcMMAE control antibody-drug conjugate, huYW353-vcMMAEantibody-drug conjugate, mu8E11-vcMMAE antibody-drug conjugate,mu2H6-vcMMAE antibody-drug conjugate, or mu3G12-vcMMAE antibody-drugconjugate; or with vehicle (PBS) alone. The presence of the antibodieswas confirmed by PK bleeds one day post injection.

As shown in FIG. 13, substantial tumor growth inhibition was achievedwith all four anti-LgR5 antibody-drug conjugates tested.

M. Efficacy of Anti-LgR5 Antibody Drug Conjugates in D5124 PancreaticCancer Xenograft

The efficacy of the anti-LgR5 ADCs was investigated using a D5124pancreatic cancer xenograft model, which has a β-catenin mutation. LgR5is highly expressed in D5124 tumors, and was confirmed by microarray,TaqMan® quantitative RT-PCR, in situ hybridization, FACS, and Westernblot. Twenty to 30 mm³ D5124 tumor fragments (LgR5-positive by FACSusing YW353 and 8E11) were implanted subcutaneously into the dorsalflank area of NCR nude mice and 18 days post-transplantation the micewere given a single intravenous injection of 6 mg/kg human anti-gD5B6-vcMMAE control antibody-drug conjugate, 3 mg/kg or 6 mg/kghuYW353-vcMMAE antibody-drug conjugate, 3 mg/kg or 6 mg/kg ch8E11-vcMMAEantibody-drug conjugate, 3 mg/kg or 6 mg/kg ch2H6-vcMMAE antibody-drugconjugate, or 3 mg/kg or 6 mg/kg ch3G12-vcMMAE antibody-drug conjugate;or with vehicle (histidine buffer: 20 mM histidine acetate, 240 mMsucrose, 0.02% Tween 20, pH5.5; “HB#8” in FIG. 14) alone. The presenceof the antibodies was confirmed by PK bleeds one and eight days postinjection.

As shown in FIG. 14, substantial tumor growth inhibition was achieved atboth doses of huYW353-vcMMAE, ch8E11-vcMMAE, and ch3G12-vcMMAE.Substantial tumor growth inhibition was also achieved at 6 mg/kgch2H6-vcMMAE.

The efficacy of various doses of YW353-vcMMAE was then tested in theD5124 pancreatic cancer xenograft model described above. Twenty to 30mm³ D5124 tumor fragments (LgR5-positive by FACS using YW353 and 8E11)were implanted subcutaneously into the dorsal flank area of NCR nudemice and 23 days post-transplantation mice were given a singleintravenous injection of 0.5 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 12mg/kg huYW353-vcMMAE antibody-drug conjugate; or 12 mg/kg huYW353; or7.2 mg/kg or 14.4 mg/kg human anti-gD 5B6-vcMMAE control antibody-drugconjugate; or vehicle (histidine buffer: 20 mM histidine acetate, 240 mMsucrose, 0.02% Tween 20, pH5.5; “HB#8” in FIG. 15) alone. The presenceof the antibodies was confirmed by PK bleeds one, four, and 14 days postinjection.

As shown in FIG. 15, substantial tumor growth inhibition was achieved at3 mg/kg huYW353-vcMMAE, and almost complete tumor growth inhibition wasachieved at 6 mg/kg and 12 mg/kg huYW353-vcMMAE.

N. Efficacy of Mu8E11 and Mu8E11-vcMMAE in Murine IntestinalTumorigenesis Model

The efficacy of mu8E11 and mu8E11-vcMMAE was investigated in a murineintestinal tumorigenesis model, APC^(min/+); LSL-Kras^(G12D); VillinCre(“AKV mice”). AKV mice are the result of crossing APC^(min/+); VillinCre(“AV mice”) with LSL-Kras^(G12D) mice. While AV mice develop 0-4adenomas in the colon and 100 adenomas in the small intestine, AKV micedevelop an average of 140 adenomas in the colon and 100 adenomas in thesmall intestine (data not shown). LgR5 mRNA expression was measured innormal tissue and polyps of AV and AKV mice, and LgR5 was found to besignificantly overexpressed in polyps from both the small intestine andcolon in AKV mice (FIG. 16). To visualize expression of LgR5 in thesmall intestine and colon of AKV mice, AKV mice were crossed with micehaving a cassette containing an enhanced green fluorescent protein(EGFP) linked in frame to human diphtheria toxin receptor cDNA locatedin the Lgr5 gene. See Tian et al., Nature, 478: 255-260 (2011). The areaof EGFP expression in small intestine polyps and large intestine polypswere visualized in the AKV Lgr5^(DTR/+) mice. The results of thatexperiment are shown in FIG. 20. It was found that LgR5 expression didnot significantly differ between small intestine polyps and colonpolyps, and further, there was no correlation between tumor size andLgR5+ area. The mean LgR5+ area of the tumors was 8%, but varied widelybetween tumors. Preliminary results show that the LgR5+ area incolorectal tumors of humans may be significantly higher than in mice,suggesting that the therapeutic index of anti-LgR5 ADC therapy in humansmay be even better than in mice.

To assess Lgr5 expression differences between intestinal crypts andtumors within these animals, intestinal tracts from AKV Lgr5^(DTR/+)mice were obtained and direct visualization of GFP was performed ontissue sections. Tumors and normal crypts were thereafter quantitatedfor the intensity of each GFP positive pixel. To determine relative GFPintensity, the intensity score is divided by the GFP+ area. As shown inFIG. 23, LgR5 expression is higher in tumors than in intestinal cryptsin of AKV Lgr5^(DTR/+) mice.

An overall survival study was carried out with AKV mice to determinewhether anti-LgR5 antibody can increase survival. Ten AKV mice wereadministered 15 mg/kg mu8E11-MC-vc-PAB-MMAE; six AKV mice wereadministered 15 mg/kg mu8E11, and 9 mice were administered 15 mg/kgcontrol antibody anti-gp120-MC-vc-MMAE. The antibodies and ADCs wereadministered weekly beginning at 6 weeks of age until the mice eitherdied or were deemed moribund (as determined by standard criteria relatedto signs of severe lethargy, weight loss and anemia), in which case themice were sacrificed.

The results of the overall survival study are shown in FIG. 17. Theuntreated control data represents historical survival rates for 22 AKVmice. In that experiment, AKV mice administered either mu8E11 ormu8E11-MC-vc-PAB-MMAE had significantly longer survival times thanuntreated AKV mice or AKV mice administered a control ADC. Based onthese results, additional animals were evaluated as described above. Theresults of that experiment are shown in FIG. 19. In the largerexperiment, AKV mice administered mu8E11-MC-vc-PAB-MMAE hadsignificantly longer survival times than untreated AKV mice or AKV miceadministered a control ADC, and also had a longer survival time thanmice administered mu8E11. At the time of death, the AKV miceadministered control ADC and anti-LgR5-ADC had similar numbers and sizesof polyps, suggesting that anti-LgR5-ADC may slow the disease andthereby extend survival.

In order to determine whether anti-LgR5 antibody and/or anti-LgR5 ADCcaused apoptosis in the gastrointestinal tumors of AKV mice, thepresence of cleaved caspase 3 was measured as a function of tumor area.Formalin fixed paraffin embedded (FFPE) small intestine and colon tissuecollected at time of death were subjected to immunohistochemicalstaining for cleaved caspase 3 (Cell Signaling Technologies; Danvers,Mass., cat#9661L). Images of the stained slides were acquired by theOlympus Nanozoomer automated slide scanning platform and manuallyidentified tumor-specific areas were analyzed in the Matlab softwarepackage (Mathworks, Natick, Mass.). Positively stained area and totaltumor area were quantified. Although rare, cleaved caspase 3 was visiblein the crypts following treatment with anti-LgR5 ADC, but was notobserved in control ADC treated animals, suggesting that LgR5-expressingcells are being specifically targeted.

The results of that experiment are shown in FIG. 18. Both anti-LgR5antibody and anti-LgR5 ADC administration caused a statisticallysignificant increase in the percentage of tumor area in AKV mice thatwas positive for the presence of cleaved caspase 3, compared to controlADC-treated AKV mice.

In order to demonstrate that the apoptosis is occurring in LgR5+cells,AKV Lgr5^(DTR/+) mice were administered 15 mg/kg mu8E11-MC-vc-PAB-MMAEor 15 mg/kg control antibody anti-gp120-MC-vc-MMAE (day 1). On day 4,the mice were sacrificed and tumors from the gastrointestinal tract(small and large intestine) were visualized for expression of EGFP andcleaved caspase 3. The amount of CC3+GFP+ area per total cellular areawas then determined. As shown in FIG. 21A, anti-LgR5-ADC treated micetended to have a greater proportion of CC3+GFP+ area than controltreated mice, although not statistically significant in that experiment.FIG. 21B shows exemplary immunohistochemical staining from control ADCtreated mice (left panels) and anti-LgR5-ADC treated mice (rightpanels). These results demonstrate a trend towards increased apoptosisin LgR5-expressing cells upon anti-LgR5-ADC treatment.

To determine whether cell proliferation of LgR5 expressing cells isaffected by anti-LgR5 treatment, the Ki67+ area per cellular area wasmeasured in the EGFP+ cell population and EGFP− cell population from thegastrointestinal tract of control-ADC treated and anti-LgR5-ADC treatedAKV Lgr5^(DTR/+) mice. Ki67 is a nuclear protein associated withcellular proliferation. Ki67 antibodies for immunohistochemical stainingwere obtained from Neomarker. The results of that experiment are shownin FIG. 22. There was significantly less proliferating cell area, asmeasured by Ki67 staining, in tumors from AKV Lgr5^(DTR/+) mice treatedwith anti-LgR5-ADC than control ADC, in both the GFP+ and GFP− cellpopulations. These results suggest that anti-LgR5-ADC reducesproliferation and/or inhibits formation of proliferative progeny.

O. Efficacy of Anti-LgR5 Antibody Drug Conjugates in D5124 PancreaticCancer Xenograft

The efficacy of the anti-LgR5 ADCs was investigated using a D5124pancreatic cancer xenograft model, which has a β-catenin mutation. LgR5is highly expressed in D5124 tumors, and was confirmed by microarray,TaqMan® quantitative RT-PCR, in situ hybridization, FACS, and Westernblot. Twenty to 30 mm³ D5124 tumor fragments were implantedsubcutaneously into the dorsal flank area of NCR nude mice and 22 dayspost-transplantation the mice were given a single intravenous injectionof 2.62 mg/kg or 5.23 mg/kg huYW353-vcMMAE antibody-drug conjugate, 3mg/kg or 6 mg/kg ch8E11-vcMMAE antibody-drug conjugate, or 3 mg/kg or 6mg/kg hu8E11v2-vcMMAE antibody-drug conjugate; or 6 mg/kg humanizedanti-gD 5B6-vcMMAE control antibody-drug conjugate; or with vehicle(histidine buffer: 20 mM histidine acetate, 240 mM sucrose, 0.02% Tween20, pH5.5) alone (n=8 mice per group). The presence of the antibodieswas confirmed by PK bleeds one and eight days post injection.

As shown in FIG. 24, substantial tumor growth inhibition was achieved atboth doses of huYW353-vcMMAE, both doses of hu8E11v2-vcMMAE, and 6 mg/kgch8E11v2-vcMMAE.

The efficacy of various doses of hu8E11v2-vcMMAE antibody-drug conjugatewas then tested in the D5124 pancreatic cancer xenograft model describedabove. Twenty to 30 mm³ D5124 tumor fragments were implantedsubcutaneously into the dorsal flank area of NCR nude mice and 26 dayspost-transplantation mice were given a single intravenous injection of0.5 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 12 mg/kg hu8E11v2-vcMMAEantibody-drug conjugate; or 15 mg/kg hu8E11v2; or 6.37 mg/kg or 12.73mg/kg human anti-gD 5B6-vcMMAE control antibody-drug conjugate; or 15mg/kg humanized anti-gD control antibody; or vehicle (histidine buffer:20 mM histidine acetate, 240 mM sucrose, 0.02% Tween 20, pH5.5) alone(n=8 mice per group).

As shown in FIG. 25, substantial tumor growth inhibition was achieved at3 mg/kg and 6 mg/kg hu8E11v2-vcMMAE, and tumor regression was achievedat 12 mg/kg hu8E11v2-vcMMAE.

P. Efficacy of Anti-LgR5 Antibody Drug Conjugates in LoVoX1.1 ColonCancer Cell Line Xenograft

The efficacy of the anti-LgR5 ADCs was investigated using a LoVo coloncancer xenograft model. LoVo cells are a colorectal adenocarcinoma cellline with an APC mutation (ATCC # CCL 229), and subline LoVoX1.1 wasderived for optimal growth in mice. Briefly, mice were inoculated withLoVo cells. Once tumors were growing, a tumor with a desirable growthrate was harvested. The tumor was minced and grown in culture toestablish cell line LoVoX1.1. LgR5 is expressed in LoVoX1.1 cells, andwas confirmed by microarray, TaqMan® quantitative RT-PCR, in situhybridization, FACS, and Western blot. Five million LoVoX1.1 cells inHBSS-matrigel were injected subcutaneously into the dorsal flank ofC.B-17 SCIDmice and 13 days post-inoculation mice were given a singleintravenous injection of 3 mg/kg or 6 mg/kg huYW353-vcMMAE antibody-drugconjugate, 3 mg/kg or 6 mg/kg hu8E11v2-vcMMAE antibody-drug conjugate;15 mg/kg hu8E11v2 antibody; 15 mg/kg humanized anti-gD 5B6 controlantibody; or 6 mg/kg humanized anti-gD 5B6-vcMMAE control antibody-drugconjugate; or with vehicle (histidine buffer: 20 mM histidine acetate,240 mM sucrose, 0.02% Tween 20, pH5.5) alone (n=10 mice per group).

As shown in FIG. 26, substantial tumor growth inhibition was achieved at6 mg/kg hu8E11v2-vcMMAE and 6 mg/kg huYW353-vcMMAE.

The efficacy of various doses of hu8E11v2-vcMMAE antibody-drug conjugatewas then tested in the LoVoX1.1 colorectal adenocarcinoma xenograftmodel described above. Five million LoVoX1.1 cells in HBSS-matrigel wereinjected subcutaneously into the dorsal flank of C.B-17 SCID mice and 10days post-inoculation mice were given a single intravenous injection of1 mg/kg, 3 mg/kg, 6 mg/kg, 10 mg/kg, or 15 mg/kg hu8E11v2-vcMMAEantibody-drug conjugate; or 15 mg/kg hu8E11v2; or 6 mg/kg or 15 mg/kghumanized anti-gD 5B6-vcMMAE control antibody-drug conjugate; or vehicle(histidine buffer: 20 mM histidine acetate, 240 mM sucrose, 0.02% Tween20, pH5.5) alone (n=9 mice per group). The presence of the antibodieswas confirmed by PK bleeds one, seven, and 14 days post injection.

As shown in FIG. 27, substantial tumor growth inhibition was achieved at6 mg/kg, 10 mg/kg, and 15 mg/kg hu8E11v2-vcMMAE.

Q. Efficacy of Anti-LgR5 Antibody Drug Conjugates in D5124 PancreaticCancer Xenograft

The efficacy of the anti-LgR5 ADCs was investigated using a D5124pancreatic cancer xenograft model, which has a β-catenin mutation. LgR5is highly expressed in D5124 tumors, and was confirmed by microarray,TaqMan® quantitative RT-PCR, in situ hybridization, FACS, and Westernblot. Twenty to 30 mm³ D5124 tumor fragments were implantedsubcutaneously into the dorsal flank area of NCR nude mice and 26 dayspost-transplantation the mice were given a single intravenous injectionof 1 mg/kg huYW353-vcMMAE antibody-drug conjugate, 1 mg/kg huYW353-vcPNUantibody-drug conjugate, 1 mg/kg huYW353-PNU antibody-drug conjugate, 1mg/kg huYW353-acetal-PNU antibody-drug conjugate; or 1 mg/kg humanizedanti-gD 5B6-vcPNU control antibody-drug conjugate, 1 mg/kg humanizedanti-gD 5B6-PNU control antibody-drug conjugate, or 1 mg/kg humanizedanti-gD 5B6-acetal-PNU control antibody-drug conjugate; or with vehicle(histidine buffer: 20 mM histidine acetate, 240 mM sucrose, 0.02% Tween20, pH5.5) alone (n=9 mice per group). The presence of the antibodieswas confirmed by PK bleeds three, seven, and 14 days post injection.

As shown in FIG. 28, substantial tumor growth inhibition was achievedwith 1 mg/kg huYW353-vcMMAE and 1 mg/kg huYW353-acetal-PNU, and almostcomplete tumor growth inhibition was achieved with 1 mg/kghuYW353-vcPNU. One of the mice treated with 1 mg/kg huYW353-acetal-PNUshowed a complete response (i.e., the mouse had no detectable tumor atthe end of the study). In addition, two of the mice treated with 1 mg/kghuYW353-vcPNU showed a partial response (i.e., >50% reduction of theinitial tumor volume at day 0).

R. Efficacy of Anti-LgR5 Antibody Drug Conjugates in D5124 PancreaticCancer Xenograft

The efficacy of various doses of hu8E11v2 antibody-drug conjugate wastested in the D5124 pancreatic cancer xenograft model. Twenty to 30 mm³D5124 tumor fragments were implanted subcutaneously into the dorsalflank area of NCR nude mice and 22 days post-transplantation mice weregiven a single intravenous injection of 2 mg/kg hu8E11v2-vcMMAEantibody-drug conjugate; or 2 mg/kg hu8E11v2-vcPNU; or 2 mg/kg or 10mg/kg hu8E11v2-acetal-PNU; or 2 mg/kg or 10 mg/kg hu8E11v2-PNU; 2 mg/kghumanized anti-gD 5B6-vcPNU control antibody-drug conjugate, 10 mg/kghumanized anti-gD 5B6-acetal-PNU control antibody drug conjugate, or 10mg/kg humanized anti-gD 5B6-PNU control antibody drug conjugate; orvehicle (histidine buffer: 20 mM histidine acetate, 240 mM sucrose,0.02% Tween 20, pH5.5) alone (n=8 mice per group).

As shown in FIG. 29, substantial tumor growth inhibition was achieved at2 mg/kg hu8E11v2-acetal-PNU, and almost complete tumor growth inhibitionwas achieved at 10 mg/kg hu8E11v2-acetal-PNU, 2 mg/kg hu8E11v2-vcPNU,and 2 mg/kg and 10 mg/kg hu8E11v2-PNU.

S. Efficacy of Anti-LgR5 Antibody Drug Conjugates in LoVo Colon CancerCell Line Xenograft

The efficacy of the anti-LgR5 ADCs was investigated using a LoVo coloncancer xenograft model. LoVo cells are a colorectal adenocarcinoma cellline with an APC mutation (ATCC #CCL 229), and subline LoVoX1.1 wasderived for optimal growth in mice. Briefly, mice were inoculated withLoVo cells. Once tumors were growing, a tumor with a desirable growthrate was harvested. The tumor was minced and grown in culture toestablish cell line LoVoX1.1. LgR5 is expressed in LoVoX1.1 cells, andwas confirmed by microarray, TaqMan® quantitative RT-PCR, in situhybridization, FACS, and Western blot. Five million LoVoX1.1 cells inHBSS-matrigel were injected subcutaneously into the dorsal flank ofC.B-17 SCID mice and 11 days post-inoculation mice were given a singleintravenous injection of 2 mg/kg hu8E11v2-vcMMAE antibody-drugconjugate, 2 mg/kg hu8E11v2-vcPNU, 2 mg/kg hu8E11v2-acetal-PNU, or 2mg/kg hu8E11v2-PNU; or 2 mg/kg humanized anti-gD 5B6-vcPNU controlantibody-drug conjugate, 2 mg/kg humanized anti-gD 5B6-acetal-PNUcontrol antibody drug conjugate, or 2 mg/kg humanized anti-gD 5B6-PNUcontrol antibody drug conjugate; or with vehicle (histidine buffer: 20mM histidine acetate, 240 mM sucrose, 0.02% Tween 20, pH5.5) alone (n=10mice per group). The presence of the antibodies was confirmed by PKbleeds one, seven, and 14 days post injection.

As shown in FIG. 30, in this experiment, certain control antibodiesappeared to show substantial tumor growth inhibition (see 2 mg/kghumanized anti-gD 5B6-vcPNU and 2 mg/kg humanized anti-gD 5B6-acetal-PNUcontrol antibody drug conjugate).

Because of the apparent non-specific effects of the control antibody inthe prior experiment, the LoVoX1.1 colorectal adenocarcinoma model wastested with a different control antibody-drug conjugate (that binds to adifferent antigen not expressed on the surface of LoVo cells), and withadministration of an excess of anti-gD control antibody to blockpossible nonspecific antibody binding sites on the tumor cells. Fivemillion LoVoX1.1 cells in HBSS-matrigel were injected subcutaneouslyinto the dorsal flank of C.B-17 SCID mice and seven dayspost-inoculation mice were given a single intravenous injection of 10mg/kg hu8E11v2-acetal-PNU antibody-drug conjugate; or 10 mg/kgthioAb-acetal-PNU control antibody-drug conjugate; or vehicle (50 mMsodium phosphate, 240 mM sucrose, 0.02% Tween20, pH 7) alone (n=5 miceper group). In addition, the mice were administered 30 mg/kg humanizedanti-gD control antibody i.p. once per week until the end of the study,beginning on the same day as, but 4 hours prior to, administration ofthe test antibodies.

As shown in FIG. 31, substantial tumor growth inhibition was achievedwith 10 mg/kg hu8E11v2-acetal-PNU and the control antibody did notinhibit tumor growth.

T. Efficacy of Anti-LgR5 Antibody Drug Conjugates in D5124 PancreaticCancer Xenograft

The efficacy of the YW353 anti-LgR5 ADCs was investigated using theD5124 pancreatic cancer xenograft model, which has β-catenin mutation.LgR5 is highly expressed in D5124 tumors, and was confirmed bymicroarray, TaqMan® quantitative RT-PCR, in situ hybridization, FACS,and Western blot. Twenty to 30 mm³ D5124 tumor fragments were implantedsubcutaneously into the dorsal flank area of NCR nude mice and 26 dayspost-transplantation the mice were given a single intravenous injectionof 1 mg/kg huYW353-vcMMAE antibody-drug conjugate, 1 mg/kg huYW353-vcPBDantibody-drug conjugate; or 1 mg/kg humanized anti-gD 5B6-vcPBD controlantibody-drug conjugate; or with vehicle (histidine buffer: 20 mMhistidine acetate, 240 mM sucrose, 0.02% Tween 20, pH5.5) alone (n=9mice per group). The presence of the antibodies was confirmed by PKbleeds one, seven, and fourteen days post injection.

As shown in FIG. 32, substantial tumor growth inhibition was achievedwith 1 mg/kg huYW353-vcMMAE and 1 mg/kg huYW353-vcPBD. One of the micetreated with 1 mg/kg huYW353-vcPBD showed a complete response (i.e., themouse had no detectable tumor at the end of the study).

In a separate experiment, efficacy of the hu8E11v2 anti-LgR5 ADCs wasinvestigated using the D5124 pancreatic cancer xenograft model. Twentyto 30 mm³ D5124 tumor fragments were implanted subcutaneously into thedorsal flank area of NCR nude mice and 22 days post-transplantation themice were given a single intravenous injection of 2 mg/kghu8E11v2-vcMMAE antibody-drug conjugate, 2 mg/kg hu8E11v2-vcPBDantibody-drug conjugate; or 2 mg/kg humanized anti-gD 5B6-vcPBD controlantibody-drug conjugate; or with vehicle (histidine buffer: 20 mMhistidine acetate, 240 mM sucrose, 0.02% Tween 20, pH5.5) alone (n=8mice per group).

As shown in FIG. 33, substantial tumor growth inhibition was achievedwith 2 mg/kg hu8E11v2-vcMMAE, and tumor regression was achieved with 2mg/kg hu8E11v2-vcPBD.

U. Efficacy of Anti-LgR5 Antibody Drug Conjugates in LoVoX1.1 ColonCancer Cell Line Xenograft

The efficacy of the anti-LgR5 ADCs was investigated using a LoVoX1.1colon cancer xenograft model. LoVo cells are a colorectal adenocarcinomacell line with an APC mutation (ATCC #CCL 229), and subline LoVoX1.1 wasderived for optimal growth in mice. Briefly, mice were inoculated withLoVo cells. Once tumors were growing, a tumor with a desirable growthrate was harvested. The tumor was minced and grown in culture toestablish cell line LoVoX1.1. LgR5 is expressed in LoVoX1.1 cells, andwas confirmed by microarray, TaqMan® quantitative RT-PCR, in situhybridization, FACS, and Western blot. Five million LoVoX1.1 cells inHBSS-matrigel were injected subcutaneously into the dorsal flank ofC.B-17 SCID mice and 11 days post-inoculation mice were given a singleintravenous injection of 2 mg/kg hu8E11v2-vcMMAE antibody-drugconjugate, 2 mg/kg hu8E11v2-vcPBD antibody-drug conjugate; or 2 mg/kghumanized anti-gD 5B6-vcPBD control antibody-drug conjugate; or withvehicle (histidine buffer: 20 mM histidine acetate, 240 mM sucrose,0.02% Tween 20, pH5.5) alone (n=10 mice per group). The presence of theantibodies was confirmed by PK bleeds one, seven, and 14 days postinjection.

As shown in FIG. 34, complete tumor growth inhibition was achieved with2 mg/kg hu8E11v2-vcPBD.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

Table of Sequences SEQ ID NO Description Sequence 1 huK_(IV)DIVMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLAWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLTISSLQAEDVA VYYCQQYYST PFTFGQGTKV EIKR 2 huVH₁EVQLVQSGAE VKKPGASVKV SCKASGYTFT SYYIHWVRQAPGQGLEWIGW INPGSGNTNY AQKFQGRVTI TRDTSTSTAYLELSSLRSED TAVYYCARFD YWGQGTLVTV SS 3 mu8E11 lightNIVLTQSPAS LAVSLGQRAT ISCRASESVD NYGNSFMHWY chain variableQQKPGQPPKL LIYLASNLES GVPARFSGSG SRTDFTLTID regionPVEADDAATY YCQQNYEDPF TFGSGTKVEI KR 4 mu8E11 heavyQVQLQQSGTE LMKPGASVKI SCKATGYTFS AYWIEWIKQR chain variablePGHGLEWIGE ILPGSDSTDY NEKFKVKATF SSDTSSNTVY regionIQLNSLTYED SAVYYCARGG HYGSLDYWGQ GTTLKVSS 5 hu8E11.v1 lightDIVMTQSPDS LAVSLGERAT INCRASESVD NYGNSFMHWY chain variableQQKPGQPPKL LIYLASNLES GVPDRFSGSG SGTDFTLTIS regionSLQAEDVAVY YCQQNYEDPF TFGQGTKVEI KR 6 hu8E11.v1 heavyEVQLVQSGAE VKKPGASVKV SCKASGYTFS AYWIEWVRQA chain variablePGQGLEWIGE ILPGSDSTDY NEKFKVRVTI TSDTSTSTVY regionLELSSLRSED TAVYYCARGG HYGSLDYWGQ GTLVTVSS 7 hu8E11.v2 lightDIVMTQSPDS LAVSLGERAT INCRASESVD NYGNSFMHWY chain variableQQKPGQPPKL LIYLASNLES GVPDRFSGSG SGTDFTLTIS regionSLQAEDVAVY YCQQNYEDPF TFGQGTKVEI KR 8 hu8E11.v2 heavyEVQLVQSGAE VKKPGASVKV SCKASGYTFS AYWIEWVRQA chain variablePGQGLEWIGE ILPGSDSTDY NEKFKVRATF TSDTSTSTVY regionLELSSLRSED TAVYYCARGG HYGSLDYWGQ GTLVTVSS 9 hu8E11.v3 lightDIVMTQSPDS LAVSLGERAT INCRASESVD NYGNSFMHWY chain variableQQKPGQPPKL LIYLASNLES GVPDRFSGSG SRTDFTLTIS regionSLQAEDVAVY YCQQNYEDPF TFGQGTKVEI KR 10 hu8E11.v3 heavyEVQLVQSGAE VKKPGASVKV SCKASGYTFS AYWIEWVRQA chain variablePGQGLEWIGE ILPGSDSTDY NEKFKVRVTI TSDTSTSTVY regionLELSSLRSED TAVYYCARGG HYGSLDYWGQ GTLVTVSS 11 hu8E11.v4 lightDIVMTQSPDS LAVSLGERAT INCRASESVD NYGNSFMHWY chain variableQQKPGQPPKL LIYLASNLES GVPDRFSGSG SRTDFTLTIS regionSLQAEDVAVY YCQQNYEDPF TFGQGTKVEI KR 12 hu8E11.v4 heavyEVQLVQSGAE VKKPGASVKV SCKASGYTFS AYWIEWVRQA chain variablePGQGLEWIGE ILPGSDSTDY NEKFKVRATF TSDTSTSTVY regionLELSSLRSED TAVYYCARGG HYGSLDYWGQ GTLVTVSS 13 hu8E11.v5 lightDIVMTQSPDS LAVSLGERAT INCRASESVD NYGNSFMHWY chain variableQQKPGQPPKL LIYLASNLES GVPDRFSGSG SGTDFTLTIS regionSLQAEDVAVY YCQQNYEDPF TFGQGTKVEI KR 14 hu8E11.v5 heavyEVQLVQSGAE VKKPGASVKV SCKASGYTFS AYWIEWVRQA chain variablePGQGLEWIGE ILPGSDSTDY NEKFKVRVTI TRDTSTSTAY regionLELSSLRSED TAVYYCARGG HYGSLDYWGQ GTLVTVSS 15 hu8E11.v6 lightDIVMTQSPDS LAVSLGERAT INCRASESVD NYGNSFMHWY chain variableQQKPGQPPKL LIYLASNLES GVPDRFSGSG SGTDFTLTIS regionSLQAEDVAVY YCQQNYEDPF TFGQGTKVEI KR 16 hu8E11.v6 heavyEVQLVQSGAE VKKPGASVKV SCKASGYTFS AYWIEWVRQA chain variablePGQGLEWIGE ILPGSDSTDY NEKFKVRVTI TADTSTSTAY regionLELSSLRSED TAVYYCARGG HYGSLDYWGQ GTLVTVSS 17 hu8E11.v7 lightDIVMTQSPDS LAVSLGERAT INCRASESVD NYGNSFMHWY chain variableQQKPGQPPKL LIYLASNLES GVPDRFSGSG SRTDFTLTIS regionSLQAEDVAVY YCQQNYEDPF TFGQGTKVEI KR 18 hu8E11.v7 heavyEVQLVQSGAE VKKPGASVKV SCKASGYTFS AYWIEWVRQA chain variablePGQGLEWIGE ILPGSDSTDY NEKFKVRVTI TRDTSTSTAY regionLELSSLRSED TAVYYCARGG HYGSLDYWGQ GTLVTVSS 19 hu8E11.v8 lightDIVMTQSPDS LAVSLGERAT INCRASESVD NYGNSFMHWY chain variableQQKPGQPPKL LIYLASNLES GVPDRFSGSG SRTDFTLTIS regionSLQAEDVAVY YCQQNYEDPF TFGQGTKVEI KR 20 hu8E11.v8 heavyEVQLVQSGAE VKKPGASVKV SCKASGYTFS AYWIEWVRQA chain variablePGQGLEWIGE ILPGSDSTDY NEKFKVRVTI TADTSTSTAY regionLELSSLRSED TAVYYCARGG HYGSLDYWGQ GTLVTVSS 21 mu3G12 lightDVVMTQTPLS LPVSLGDQAS ISCRSSQSLV HSNGNTYLQW chain variableYLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI regionSRVEAEDLGI YFCSQSTHFP YTFGGGTKLE IKR 22 mu3G12 heavyQVQLQQPGAE MVKPGASVKL SCKASVDTFN SYWMHWVKQR chain variablePGQGLEWIGE INPSNGRTNY IEKFKNRATV TVDKSSSTAF regionMQLSSLTSED SAVYYCATGW YFDVWGAGTT VTVSS 23 mu2H6 light chainDIVMTQSPSS LTVTAGEKVT MSCKSSQSLL NSGNQKNYLT variable regionWFQQKPGQPP KLLIYWASTR ESGVPDRFTG SGSGTDFTLTISNVQAEDLA VYYCQNDYSF PFTFGQGTKV EIKR 24 mu2H6 heavyEVQLQQSGPE LVKPGTSMKI SCKASGYSFT GYTMNWVKQS chain variableHKNGLEWIGL INCYNGGTNY NQKFKGKATL TVDKSSSTAF regionMELLSLTSED SAVYYCARGG STMITPRFAY WGQGTLVTVS S 25 YW353 lightDIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP chain variableGKAPKLLIYS ASFLYSGVPS RFSGSGSGTD FTLTISSLQP regionEDFATYYCQQ SYTTPPTFGQ GTKVEIKR 26 YW353 heavyEVQLVESGGG LVQPGGSLRL SCAASGFTFT SYSISWVRQA chain variablePGKGLEWVAE IYPPGGYTDY ADSVKGRFTI SADTSKNTAY regionLQMNSLRAED TAVYYCAKAR LFFDYWGQGT LVTVSS 27 nm8E11 HVR L1RASESVDNYG NSFMH 28 mu8E11 HVR L2 LASNLES 29 mu8E11 HVR L3 QQNYEDPFT 30mu8E11 HVR H1 GYTFSAYWIE 31 mu8E11 HVR H2 EILPGSDSTD YNEKFKV 32mu8E11 HVR H3 GGHYGSLDY 33 Hu8E11 light DIVMTQSPDS LAVSLGERAT INCchain (LC) framework 1 (FR1) 34 Hu8E11 LC FR2 WYQQKPGQPP KLLIY 35Hu8E11.v1 LC GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YC FR3 Hu8E11.v2 LC FR3Hu8E11.v5 LC FR3 Hu8E11.v6 LC FR3 36 Hu8E11.v3 LCGVPDRFSGSG SRTDFTLTIS SLQAEDVAVY YC FR3 Hu8E11.v4 LC FR3 Hu8E11.v7 LCFR3 Hu8E11.v8 LC FR3 37 Hu8E11 LC FR4 FGQGTKVEIK R 38 Hu8E11 heavyEVQLVQSGAE VKKPGASVKV SCKAS chain (HC) framework1 (FR1) 39 Hu8E11 HC FR2WVRQAPGQGL EWIG 40 Hu8E11.v1 HC RVTITSDTST STVYLELSSL RSEDTAVYYC AR FR3Hu8E11.v3 HC FR3 41 Hu8E11.v2 HC RATFTSDTST STVYLELSSL RSEDTAVYYC AR FR3Hu8E11.v4 HC FR3 42 Hu8E11.v5 HC RVTITRDTST STAYLELSSL RSEDTAVYYC AR FR3Hu8E11.v7 HC FR3 43 Hu8E11.v6 HC RVTITADTST STAYLELSSL RSEDTAVYYC AR FR3Hu8E11.v8 HC FR3 44 Hu8E11 HC FR4 WGQGTLVTVS S 45 mu3G12 HVR L1RSSQSLVHSN GNTYLQ 46 mu3G12 HVR L2 KVSNRFS 47 mu3G12 HVR L3 SQSTHFPYT 48mm3G12 HVR H1 VDTFNSYWMH 49 mu3G12 HVR H2 EINPSNGRTN YIEKFKN 50mu3G12 HVR H3 GWYFDV 51 mu2H6 HVR L1 KSSQSLLNSG NQKNYLT 52 mu2H6 HVR L2WASTRES 53 mu2H6 HVR L3 QNDYSFPFT 54 mu2H6 HVR H1 GYSFTGYTMN 55mu2H6 HVR H2 LINCYNGGTN YNQKFKG 56 mu2H6 HVR H3 GGSTMITPRF AY 57YW353 HVR L1 RASQDVSTAV A 58 YW353 HVR L2 SASFLYS 59 YW353 HVR L3QQSYTTPPT 60 YW353 HVR H1 GFTFTSYSIS 61 YW353 HVR H2 EIYPPGGYTD YADSVKG62 YW353 HVR H3 ARLFFDY 63 hu8E11.v2 lightDIVMTQSPDS LAVSLGERAT INCRASESVD NYGNSFMHWY chainQQKPGQPPKL LIYLASNLES GVPDRFSGSG SGTDFTLTISSLQAEDVAVY YCQQNYEDPF TFGQGTKVEI KRTVAAPSVFIFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQSGNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 64hu8E11.v2 heavy EVQLVQSGAE VKKPGASVKV SCKASGYTFS AYWIEWVRQA chainPGQGLEWIGE ILPGSDSTDY NEKFKVRATF TSDTSTSTVYLELSSLRSED TAVYYCARGG HYGSLDYWGQ GTLVTVSSASTKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYICNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYVDGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEYKCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMTKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 65 YW353 lightDIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP chainGKAPKLLIYS ASFLYSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ SYTTPPTFGQ GTKVEIKRTV AAPSVFIFPPSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 66YW353 heavy EVQLVESGGG LVQPGGSLRL SCAASGFTFT SYSISWVRQA chainPGKGLEWVAE IYPPGGYTDY ADSVKGRFTI SADTSKNTAYLQMNSLRAED TAVYYCAKAR LFFDYWGQGT LVTVSSASTKGPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSGALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVFLFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDGVEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKCKVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKNQVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK 67 Human LgR5MDTSRLGVLL SLPVLLQLAT GGSSPRSGVL LRGCPTHCHC precursor;EPDGRMLLRV DCSDLGLSEL PSNLSVFTSY LDLSMNNISQ LGR5_humanLLPNPLPSLR FLEELRLAGN ALTYIPKGAF TGLYSLKVLM NP_003658;LQNNQLRHVP TEALQNLRSL QSLRLDANHI SYVPPSCFSG signal sequence =LHSLRHLWLD DNALTEIPVQ AFRSLSALQA MTLALNKIHH amino acids 1-21IPDYAFGNLS SLVVLHLHNN RIHSLGKKCF DGLHSLETLDLNYNNLDEFP TAIRTLSNLK ELGFHSNNIR SIPEKAFVGNPSLITIHFYD NPIQFVGRSA FQHLPELRTL TLNGASQITEFPDLTGTANL ESLTLTGAQI SSLPQTVCNQ LPNLQVLDLSYNLLEDLPSF SVCQKLQKID LRHNEIYEIK VDTFQQLLSLRSLNLAWNKI AIIHPNAFST LPSLIKLDLS SNLLSSFPITGLHGLTHLKL TGNHALQSLI SSENFPELKV IEMPYAYQCCAFGVCENAYK ISNQWNKGDN SSMDDLHKKD AGMFQAQDERDLEDFLLDFE EDLKALHSVQ CSPSPGPFKP CEHLLDGWLIRIGVWTIAVL ALTCNALVTS TVFRSPLYIS PIKLLIGVIAAVNMLTGVSS AVLAGVDAFT FGSFARHGAW WENGVGCHVIGFLSIFASES SVFLLTLAAL ERGFSVKYSA KFETKAPFSSLKVIILLCAL LALTMAAVPL LGGSKYGASP LCLPLPFGEPSTMGYMVALI LLNSLCFLMM TIAYTKLYCN LDKGDLENIWDCSMVKHIAL LLFTNCILNC PVAFLSFSSL INLTFISPEVIKFILLVVVP LPACLNPLLY ILFNPHFKED LVSLRKQTYVWTRSKHPSLM SINSDDVEKQ SCDSTQALVT FTSSSITYDLPPSSVPSPAY PVTESCHLSS VAFVPCL 68 Human LgR5 GSSPRSGVL LRGCPTHCHC EPDGRMLLRV DCSDLGLSEL mature, withoutPSNLSVFTSY LDLSMNNISQ LLPNPLPSLR FLEELRLAGN signal sequence;ALTYIPKGAF TGLYSLKVLM LQNNQLRHVP TEALQNLRSL amino acids 22 toQSLRLDANHI SYVPPSCFSG LHSLRHLWLD DNALTEIPVQ 907AFRSLSALQA MTLALNKIHH IPDYAFGNLS SLVVLHLHNNRIHSLGKKCF DGLHSLETLD LNYNNLDEFP TAIRTLSNLKELGFHSNNIR SIPEKAFVGN PSLITIHFYD NPIQFVGRSAFQHLPELRTL TLNGASQITE FPDLTGTANL ESLTLTGAQISSLPQTVCNQ LPNLQVLDLS YNLLEDLPSF SVCQKLQKIDLRHNEIYEIK VDTFQQLLSL RSLNLAWNKI AIIHPNAFSTLPSLIKLDLS SNLLSSFPIT GLHGLTHLKL TGNHALQSLISSENFPELKV IEMPYAYQCC AFGVCENAYK ISNQWNKGDNSSMDDLHKKD AGMFQAQDER DLEDFLLDFE EDLKALHSVQCSPSPGPFKP CEHLLDGWLI RIGVWTIAVL ALTCNALVTSTVFRSPLYIS PIKLLIGVIA AVNMLTGVSS AVLAGVDAFTFGSFARHGAW WENGVGCHVI GFLSIFASES SVFLLTLAALERGFSVKYSA KFETKAPFSS LKVIILLCAL LALTMAAVPLLGGSKYGASP LCLPLPFGEP STMGYMVALI LLNSLCFLMMTIAYTKLYCN LDKGDLENIW DCSMVKHIAL LLFTNCILNCPVAFLSFSSL INLTFISPEV IKFILLVVVP LPACLNPLLYILFNPHFKED LVSLRKQTYV WTRSKHPSLM SINSDDVEKQSCDSTQALVT FTSSSITYDL PPSSVPSPAY PVTESCHLSS VAFVPCL 69 CynomolgusGCPTHCHCEP DGRMLLRVDC SDLGLSELPS NLSVFTSYLD monkey LgR5LSMNNISQLL PNPLPSLRFL EELRLAGNAL TYIPKGAFTG partial sequence,LYSLKVLMLQ NNQLRQVPTE ALQNLRSLQS LRLDANHISY predicted;VPPSCFSGLH SLRHLWLDDN ALTEIPVQAF RSLSALQAMT predicted toLALNKIHHIP DYAFGNLSSL VVLHLHNNRI HSLGKKCFDG correspond toLHSLETLDLN YNNLDEFPTA IRTLSNLKEL GFHSNNIRSI amino acids 33 toPEKAFVGNPS LITIHFYDNP IQFVGRSAFQ HLPELRTLTL 907 of full-lengthNGASQITEFP DLTGTANLES LTLTGAQISS LPQTVCNQLP precursorNLQVLDLSYN LLEDLPSFSV CQKLQKIDLR HNEIYEIKVDTFQQLLSLRS LNLAWNKIAI IHPNAFSTLP SLIKLDLSSNLLSSFPVTGL HGLTHLKLTG NHALQSLISS ENFPELKIIEMPYAYQCCAF GVCENAYKIS NQWNKGDNSS MDDLHKKDAGMFQVQDERDL EDFLLDFEED LKALHSVQCS PSPGPFKPCEHLLDGWLIRI GVWTIAVLAL TCNALVTSTV FRSPLYISPIKLLIGVIAVV NMLTGVSSAV LAGVDAFTFG SFARHGAWWENGVGCQVIGF LSIFASESSV FLLTLAALER GFSVKCSAKFETKAPFSSLK VIILLCALLA LTMAAVPLLG GSEYGASPLCLPLPFGEPST TGYMVALILL NSLCFLMMTI AYTKLYCNLDKGDLENIWDC SMVKHIALLL FTNCILYCPV AFLSFSSLLNLTFISPEVIK FILLVIVPLP ACLNPLLYIL FNPHFKEDLVSLGKQTYFWT RSKHPSLMSI NSDDVEKQSC DSTQALVTFTSSSIAYDLPP SSVPSPAYPV TESCHLSSVA FVPCL 70 Rat LgR5MDTSRVRMLL SLLALLQLVA AGSPPRPDTM PRGCPSYCHC precursor;ELDGRMLLRV DCSDLGLSEL PSNLSVFTSY LDLSMNNISQ LGR5_ratLPASLLHRLR FLEELRLAGN ALTHIPKGAF AGLHSLKVLM NP_001100254;LQNNQLRQVP EEALQNLRSL QSLRLDANHI SYVPPSCFSG signal sequence =LHSLRHLWLD DNALTDVPVQ AFRSLSALQA MTLALNKIHH amino acids 1-21IADHAFGNLS SLVVLHLHNN RIHSLGKKCF DGLHSLETLDLNYNNLDEFP TAIKTLSNLK ELGFHSNNIR SIPERAFVGNPSLITIHFYD NPIQFVGISA FQHLPELRTL TLNGASQITEFPDLTGTATL ESLTLTGAKI SSLPQTVCDQ LPNLQVLDLSYNLLEDLPSL SGCQKLQKID LRHNEIYEIK GGTFQQLFNLRSLNLARNKI AIIHPNAFST LPSLIKLDLS SNLLSSFPVTGLHGLTHLKL TGNRALQSLI PSANFPELKI IEMPYAYQCCAFGGCENVYK IPNQWNKDDS SSVDDLRKKD AGLFQVQDERDLEDFLLDFE EDLKVLHSVQ CSPPPGPFKP CEHLFGSWLIRIGVWTTAVL ALSCNALVAF TVFRTPLYIS SIKLLIGVIAVVDILMGVSS AILAVVDTFT FGSFAQHGAW WEGGIGCQIVGFLSIFASES SVFLLTLAAL ERGFSVKCSS KFEMKAPLSSLKAIILLCVL LALTIATVPL LGGSEYNASP LCLPLPFGEPSTTGYMVALV LLNSLCFLIM TIAYTRLYCS LEKGELENLWDCSMVKHTAL LLFTNCILYC PVAFLSFSSL LNLTFISPEVIKFILLVIVP LPACLNPLLY IVFNPHFKED MGSLGKQTRFWTRAKHPSLL SINSDDVEKR SCDSTQALVS FTHASIAYDLPSDSGSSPAY PMTESCHLSS VAFVPCL 71 Rat LgR5 mature, GSPPRPDTM PRGCPSYCHC ELDGRMLLRV DCSDLGLSEL without signalPSNLSVFTSY LDLSMNNISQ LPASLLHRLR FLEELRLAGN sequence; aminoALTHIPKGAF AGLHSLKVLM LQNNQLRQVP EEALQNLRSL acids 22 to 907QSLRLDANHI SYVPPSCFSG LHSLRHLWLD DNALTDVPVQAFRSLSALQA MTLALNKIHH IADHAFGNLS SLVVLHLHNNRIHSLGKKCF DGLHSLETLD LNYNNLDEFP TAIKTLSNLKELGFHSNNIR SIPERAFVGN PSLITIHFYD NPIQFVGISAFQHLPELRTL TLNGASQITE FPDLTGTATL ESLTLTGAKISSLPQTVCDQ LPNLQVLDLS YNLLEDLPSL SGCQKLQKIDLRHNEIYEIK GGTFQQLFNL RSLNLARNKI AIIHPNAFSTLPSLIKLDLS SNLLSSFPVT GLHGLTHLKL TGNRALQSLIPSANFPELKI IEMPYAYQCC AFGGCENVYK IPNQWNKDDSSSVDDLRKKD AGLFQVQDER DLEDFLLDFE EDLKVLHSVQCSPPPGPFKP CEHLFGSWLI RIGVWTTAVL ALSCNALVAFTVFRTPLYIS SIKLLIGVIA VVDILMGVSS AILAVVDTFTFGSFAQHGAW WEGGIGCQIV GFLSIFASES SVFLLTLAALERGFSVKCSS KFEMKAPLSS LKAIILLCVL LALTIATVPLLGGSEYNASP LCLPLPFGEP STTGYMVALV LLNSLCFLIMTIAYTRLYCS LEKGELENLW DCSMVKHTAL LLFTNCILYCPVAFLSFSSL LNLTFISPEV IKFILLVIVP LPACLNPLLYIVFNPHFKED MGSLGKQTRF WTRAKHPSLL SINSDDVEKRSCDSTQALVS FTHASIAYDL PSDSGSSPAY PMTESCHLSS VAFVPCL 72 Mouse LgR5MDTSCVHMLL SLLALLQLVA AGSSPGPDAI PRGCPSHCHC precursor;ELDGRMLLRV DCSDLGLSEL PSNLSVFTSY LDLSMNNISQ LGR5_mouseLPASLLHRLC FLEELRLAGN ALTHIPKGAF TGLHSLKVLM NP_034325;LQNNQLRQVP EEALQNLRSL QSLRLDANHI SYVPPSCFSG signal sequence =LHSLRHLWLD DNALTDVPVQ AFRSLSALQA MTLALNKIHH amino acids 1-21IADYAFGNLS SLVVLHLHNN RIHSLGKKCF DGLHSLETLDLNYNNLDEFP TAIKTLSNLK ELGFHSNNIR SIPERAFVGNPSLITIHFYD NPIQFVGVSA FQHLPELRTL TLNGASHITEFPHLTGTATL ESLTLTGAKI SSLPQAVCDQ LPNLQVLDLSYNLLEDLPSL SGCQKLQKID LRHNEIYEIK GSTFQQLFNLRSLNLAWNKI AIIHPNAFST LPSLIKLDLS SNLLSSFPVTGLHGLTHLKL TGNRALQSLI PSANFPELKI IEMPSAYQCCAFGGCENVYK ISNQWNKDDG NSVDDLHKKD AGLFQVQDERDLEDFLLDFE EDLKALHSVQ CSPSPGPFKP CEHLFGSWLIRIGVWTTAVL ALSCNALVAL TVFRTPLYIS SIKLLIGVIAVVDILMGVSS AVLAAVDAFT FGRFAQHGAW WEDGIGCQIVGFLSIFASES SIFLLTLAAL ERGFSVKCSS KFEVKAPLFSLRAIVLLCVL LALTIATIPL LGGSKYNASP LCLPLPFGEPSTTGYMVALV LLNSLCFLIM TIAYTKLYCS LEKGELENLWDCSMVKHIAL LLFANCILYC PVAFLSFSSL LNLTFISPDVIKFILLVIVP LPSCLNPLLY IVFNPHFKED MGSLGKHTRFWMRSKHASLL SINSDDVEKR SCESTQALVS FTHASIAYDLPSTSGASPAY PMTESCHLSS VAFVPCL 73 Mouse LgR5 GSSPGPDAI PRGCPSHCHC ELDGRMLLRV DCSDLGLSEL mature, withoutPSNLSVFTSY LDLSMNNISQ LPASLLHRLC FLEELRLAGN signal sequence;ALTHIPKGAF TGLHSLKVLM LQNNQLRQVP EEALQNLRSL amino acids 22 toQSLRLDANHI SYVPPSCFSG LHSLRHLWLD DNALTDVPVQ 907AFRSLSALQA MTLALNKIHH IADYAFGNLS SLVVLHLHNNRIHSLGKKCF DGLHSLETLD LNYNNLDEFP TAIKTLSNLKELGFHSNNIR SIPERAFVGN PSLITIHFYD NPIQFVGVSAFQHLPELRTL TLNGASHITE FPHLTGTATL ESLTLTGAKISSLPQAVCDQ LPNLQVLDLS YNLLEDLPSL SGCQKLQKIDLRHNEIYEIK GSTFQQLFNL RSLNLAWNKI AIIHPNAFSTLPSLIKLDLS SNLLSSFPVT GLHGLTHLKL TGNRALQSLIPSANFPELKI IEMPSAYQCC AFGGCENVYK ISNQWNKDDGNSVDDLHKKD AGLFQVQDER DLEDFLLDFE EDLKALHSVQCSPSPGPFKP CEHLFGSWLI RIGVWTTAVL ALSCNALVALTVFRTPLYIS SIKLLIGVIA VVDILMGVSS AVLAAVDAFTFGRFAQHGAW WEDGIGCQIV GFLSIFASES SIFLLTLAALERGFSVKCSS KFEVKAPLFS LRAIVLLCVL LALTIATIPLLGGSKYNASP LCLPLPFGEP STTGYMVALV LLNSLCFLIMTIAYTKLYCS LEKGELENLW DCSMVKHIAL LLFANCILYCPVAFLSFSSL LNLTFISPDV IKFILLVIVP LPSCLNPLLYIVFNPHFKED MGSLGKHTRF WMRSKHASLL SINSDDVEKRSCESTQALVS FTHASIAYDL PSTSGASPAY PMTESCHLSS VAFVPCL 74 hu8E11.v2 V205CDIVMTQSPDS LAVSLGERAT INCRASESVD NYGNSFMHWY cysteineQQKPGQPPKL LIYLASNLES GVPDRFSGSG SGTDFTLTIS engineered lightSLQAEDVAVY YCQQNYEDPF TFGQGTKVEI KRTVAAPSVF chain (Igκ)IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQSGNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPCT KSFNRGEC 75hu8E11.v2 A118C EVQLVQSGAE VKKPGASVKV SCKASGYTFS AYWIEWVRQA cysteinePGQGLEWIGE ILPGSDSTDY NEKFKVRATF TSDTSTSTVY engineered heavyLELSSLRSED TAVYYCARGG HYGSLDYWGQ GTLVTVSSCS chain (IgG1)TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYICNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYVDGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEYKCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMTKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 76 hu8E11.v2 S400CEVQLVQSGAE VKKPGASVKV SCKASGYTFS AYWIEWVRQA cysteinePGQGLEWIGE ILPGSDSTDY NEKFKVRATF TSDTSTSTVY engineered heavyLELSSLRSED TAVYYCARGG HYGSLDYWGQ GTLVTVSSAS chain (IgG1)TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYICNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYVDGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEYKCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMTKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDCDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 77 YW353 V205CDIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP cysteineGKAPKLLIYS ASFLYSGVPS RFSGSGSGTD FTLTISSLQP engineered lightEDFATYYCQQ SYTTPPTFGQ GTKVEIKRTV AAPSVFIFPP chain (Igκ)SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPCTKSFN RGEC 78YW353 A118C EVQLVESGGG LVQPGGSLRL SCAASGFTFT SYSISWVRQA cysteinePGKGLEWVAE IYPPGGYTDY ADSVKGRFTI SADTSKNTAY engineered heavyLQMNSLRAED TAVYYCAKAR LFFDYWGQGT LVTVSSCSTK chain (IgG1)GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSGALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVFLFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDGVEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKCKVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKNQVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK 79 YW353 S400CEVQLVESGGG LVQPGGSLRL SCAASGFTFT SYSISWVRQA cysteinePGKGLEWVAE IYPPGGYTDY ADSVKGRFTI SADTSKNTAY engineered heavyLQMNSLRAED TAVYYCAKAR LFFDYWGQGT LVTVSSASTK chain (IgG1)GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSGALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVFLFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDGVEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKCKVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKNQVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDCDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK 80 V205C cysteineTVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW engineered lightKVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK chain constantHKVYACEVTH QGLSSPCTKS FNRGEC region (Igκ) 81 A118C cysteineCSTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS engineered heavyWNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT chain constantYICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG region (IgG1)PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREEMTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 82 S400C cysteineASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS engineered heavyWNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT chain constantYICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG region (IgG1)PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREEMTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDCDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

What is claimed is:
 1. An immunoconjugate comprising an isolatedantibody that binds to LgR5, wherein the antibody comprises: a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 30, HVR-H2 comprisingthe amino acid sequence of SEQ ID NO: 31, HVR-H3 comprising the aminoacid sequence of SEQ ID NO: 32, HVR-L1 comprising the amino acidsequence of SEQ ID NO: 27, HVR-L2 comprising the amino acid sequence ofSEQ ID NO: 28, and HVR-L3 comprising the amino acid sequence of SEQ IDNO: 29; or b) HVR-H1 comprising the amino acid sequence of SEQ ID NO:60, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 61, HVR-H3comprising the amino acid sequence of SEQ ID NO: 62, HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 57, HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 58, and HVR-L3 comprising the amino acidsequence of SEQ ID NO:
 59. 2. The immunoconjugate of claim 1, whereinthe antibody is a monoclonal antibody.
 3. The immunoconjugate of claim1, wherein the antibody is a human, humanized, or chimeric antibody. 4.The immunoconjugate of claim 1, wherein the antibody is an antibodyfragment that binds LgR5.
 5. The immunoconjugate of claim 1, whereinLgR5 is human LgR5 of SEQ ID NO:
 67. 6. The immunoconjugate of claim 1,wherein the antibody comprises: a) heavy chain framework FR3 sequence ofSEQ ID NO: 40; b) heavy chain framework FR3 sequence of SEQ ID NO: 41;c) heavy chain framework FR3 sequence of SEQ ID NO: 42; or d) heavychain framework FR3 sequence of SEQ ID NO:
 43. 7. The immunoconjugate ofclaim 1, wherein the antibody comprises a light chain framework FR3sequence of SEQ ID NO:
 35. 8. The immunoconjugate of claim 1, whereinthe antibody comprises: a) a VH sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO: 6; b) a VL sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO: 5; c) a VH sequence as in (a) and a VL sequence as in (b); d) aVH sequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 8; e) a VL sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO: 7; f) a VH sequence asin (d) and a VL sequence as in (e); g) a VH sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 10; h) a VLsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 9; i) a VH sequence as in (g) and a VL sequenceas in (h); j) a VH sequence having at least 95% sequence identity to theamino acid sequence of SEQ ID NO: 12; k) a VL sequence having at least95% sequence identity to the amino acid sequence of SEQ ID NO: 11; l) aVH sequence as in (j) and a VL sequence as in (k); m) a VH sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO: 14; n) a VL sequence having at least 95% sequence identity to theamino acid sequence of SEQ ID NO: 13; o) a VH sequence as in (m) and aVL sequence as in (n); p) a VH sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO: 16; q) a VL sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO: 15; r) a VH sequence as in (p) and a VL sequence as in (q); s) aVH sequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 18; t) a VL sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO: 17; u) a VH sequenceas in (s) and a VL sequence as in (t); v) a VH sequence having at least95% sequence identity to the amino acid sequence of SEQ ID NO: 20; w) aVL sequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 19; x) a VH sequence as in (v) and a VL sequenceas in (w); y) a VH sequence having at least 95% sequence identity to theamino acid sequence of SEQ ID NO: 26; z) a VL sequence having at least95% sequence identity to the amino acid sequence of SEQ ID NO: 25; oraa) a VH sequence as in (y) and a VL sequence as in (z).
 9. Theimmunoconjugate of claim 8, comprising a VH sequence selected from SEQID NOs: 6, 8, 10, 12, 14, 16, 18, 20, and
 26. 10. The immunoconjugate ofclaim 8, comprising a VL sequence selected from SEQ ID NOs: 5, 7, 9, 11,13, 15, 17, 19, and
 25. 11. An immunoconjugate comprising: a) a VHsequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 5; b) a VHsequence of SEQ ID NO: 8 and a VL sequence of SEQ ID NO: 7; c) a VHsequence of SEQ ID NO: 10 and a VL sequence of SEQ ID NO: 9; d) a VHsequence of SEQ ID NO: 12 and a VL sequence of SEQ ID NO: 11; e) a VHsequence of SEQ ID NO: 14 and a VL sequence of SEQ ID NO: 13; f) a VHsequence of SEQ ID NO: 16 and a VL sequence of SEQ ID NO: 15; g) a VHsequence of SEQ ID NO: 18 and a VL sequence of SEQ ID NO: 17; h) a VHsequence of SEQ ID NO: 20 and a VL sequence of SEQ ID NO: 19; or i) a VHsequence of SEQ ID NO: 26 and a VL sequence of SEQ ID NO: 25; and acytotoxic agent.
 12. The immunoconjugate of claim 1, which is an IgG1,IgG2a or IgG2b antibody.
 13. The immunoconjugate of claim 1 having theformula Ab-(L-D)p, wherein: (a) Ab is the antibody; (b) L is a linker;(c) D is a drug selected from a maytansinoid, an auristatin, acalicheamicin, a pyrrolobenzodiazepine, and a nemorubicin derivative;and (d) p ranges from 1-8.
 14. The immunoconjugate of claim 13, whereinD is an auristatin.
 15. The immunoconjugate of claim 14, wherein D hasformula D_(E)

and wherein R² and R⁶ are each methyl, R³ and R⁴ are each isopropyl, R⁵is H, R⁷ is sec-butyl, each R⁸ is independently selected from CH₃,O—CH₃, OH, and H; R⁹ is H; and R¹⁸ is —C(R⁸)₂—C(R⁸)₂-aryl.
 16. Theimmunoconjugate of claim 13, wherein the drug is MMAE.
 17. Theimmunoconjugate of claim 13, wherein D is a pyrrolobenzodiazepine ofFormula A:

wherein the dotted lines indicate the optional presence of a double bondbetween C1 and C2 or C2 and C3; R² is independently selected from H, OH,═O, ═CH₂, CN, R, OR, ═CH—R^(D), ═C(R^(D))₂, O—SO₂—R, CO₂R and COR, andoptionally further selected from halo or dihalo, wherein R^(D) isindependently selected from R, CO₂R, COR, CHO, CO₂H, and halo; R⁶ and R⁹are independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo; R⁷ is independently selected from H, R, OH, OR, SH,SR, NH₂, NHR, NRR′, NO₂, Me₃Sn and halo; Q is independently selectedfrom O, S and NH; R¹¹ is either H, or R or, where Q is O, SO₃M, where Mis a metal cation; R and R′ are each independently selected fromoptionally substituted C₁₋₈ alkyl, C₃₋₈ heterocyclyl and C₅₋₂₀ arylgroups, and optionally in relation to the group NRR′, R and R′ togetherwith the nitrogen atom to which they are attached form an optionallysubstituted 4-, 5-, 6- or 7-membered heterocyclic ring; R¹², R¹⁶, R¹⁹and R¹⁷ are as defined for R², R⁶, R⁹ and R⁷ respectively; R″ is a C₃₋₁₂alkylene group, which chain may be interrupted by one or moreheteroatoms and/or aromatic rings that are optionally substituted; and Xand X′ are independently selected from O, S and N(H).
 18. Theimmunoconjugate of claim 17, wherein D has the structure:

wherein n is 0 or
 1. 19. The immunoconjugate of claim 17, wherein D hasa structure selected from:

wherein R^(E) and R^(E″) are each independently selected from H orR^(D), wherein R^(D) is independently selected from R, CO₂R, COR, CHO,CO₂H, and halo; wherein Ar¹ and Ar² are each independently optionallysubstituted C₅₋₂₀ aryl; and wherein n is 0 or
 1. 20. The immunoconjugateof claim 13, wherein D is a pyrrolobenzodiazepine of Formula B:

wherein the horizontal wavy line indicates the covalent attachment siteto the linker; R^(V1) and R^(V2) are independently selected from H,methyl, ethyl, phenyl, fluoro-substituted phenyl, and C₅₋₆ heterocyclyl;and n is 0 or
 1. 21. The immunoconjugate of claim 13, wherein D is anemorubicin derivative.
 22. The immunoconjugate of claim 21, wherein Dhas a structure selected from:


23. The immunoconjugate of claim 13, wherein the linker is cleavable bya protease.
 24. The immunoconjugate of claim 23, wherein the linkercomprises a val-cit dipeptide or a Phe-Lys dipeptide.
 25. Theimmunoconjugate of claim 13, wherein the linker is acid-labile.
 26. Theimmunoconjugate of claim 25, wherein the linker comprises hydrazone. 27.The immunoconjugate of claim 15 having the formula:

wherein S is a sulfur atom.
 28. The immunoconjugate of claim 18 havingthe formula:


29. The immunoconjugate of claim 22 having a formula selected from:

wherein R₁ and R₂ are independently selected from H and C₁-C₆ alkyl. 30.The immunoconjugate of claim 13, wherein p ranges from 2-5.
 31. Apharmaceutical formulation comprising the immunoconjugate of claim 13and a pharmaceutically acceptable carrier.